Bifunctional or bivalent antibody fragment analogue

ABSTRACT

A bispecific or bivalent double head antibody fragment, which is composed of a binding complex containing two polypeptide chains, whereby one polypeptide chain has two times a variable domain of a heavy chain (V H ) in series and the other polypeptide chain has two times a variable domain of a light chain (V L ) in series, and the binding complex contains two pairs of variable domains (V H  -A//V L  -A and V H  -B//V L  -B), wherein said double head antibody fragments have binfunctional antigen binding activity. A process for producing such an antibody fragment is disclosed. An immunoassay is also provided for, wherein the improvement is the inclusion of a bispecific or bivalent double head antibody fragment, which is composed of a binding complex containing two polypeptide chains, whereby one polypeptide chain has two times a variable domain of a heavy chain (V H ) in series and the other polypeptide chain has two times a variable domain of a light chain (V L ) in series, as a binding means.

This application is the national phase filing of International application PCT/EP96/03605, filed Aug. 14, 1996, which designated the U.S. and claims priority based on GB 95307332.7, filed in Great Britain on Oct. 16, 1995.

The invention relates to new bispecific or bivalent antibody fragment analogues, a process for preparing such antibody fragment analogues and various uses of such antibody fragment analogues.

BACKGROUND OF THE INVENTION AND PRIOR ART

1. Antibody Structure

Antibody molecules typically are Y-shaped molecules whose basic unit consist of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains. The N-terminal regions, also known as V-domains, are variable in sequence and are responsible for the antibody specificity. The antibody specifically recognizes and binds to an antigen mainly through six short complementarity-determining regions located in their V-domains (see FIG. 1).

In this specification abbreviations are used having the following meaning.

C-domain: Constant domain

V-domain: Variable domain

V.sub. : Variable domain of the light chain

V_(H) : Variable domain of the heavy chain

Fv: dual chain antibody fragment containing both a V_(H) and a V_(L)

scFv: single-chain Fv (V_(H) and V_(L) genetically linked either directly or via a peptide linker)

CDR: Complementarity-determining region

ELISA: Enzyme Linked Immuno Sorbent Assay

PCR: Polymerase Chain Reaction

IPTG: IsoPropyl-β-ThioGalactopyranoside

PBS: Phosphate Buffered Saline

PBST: Phosphate Buffered Saline with 0.15% Tween

TMB: 3,3',5,5'-TetraMethylBenzidine

It is generally known that proteolytic digestion of an antibody with papain yields three fragments. The fragment containing the CH₂ and CH₃ domains of the two heavy chains connected by the complete hinge (see FIG. 1) crystallises very easily and was therefore called Fc fragment. The two other fragments are identical and were called Fab fragments, as they contained the antigen-binding site. Digestion with pepsin is such that the two Fab's remain connected via the hinge, forming only two fragments: Fc' and Fab₂.

The Fv is the smallest unit of an antibody which still contains the complete binding site (see FIG. 1) and full antigen binding activity. It consists of only the V-domains of the heavy and light chains thus forming a small, heterodimeric variable fragment or Fv. Fv's have a molecular weight of about 25 kD, which is only one sixth of the parent whole antibody (in the case of an IgG). Previously Fv's were only available by proteolysis in a select number of cases (Givol, 1991). The production of Fv's can now be achieved more routinely using genetic engineering methods through cloning and expressing DNA encoding only the V-domains of the antibody of interest. Smaller fragments, such as individual V-domains (Domain Antibodies or dABs, Ward et al., 1989), and even individual CDR's (Williams et al., 1989; Taub et al., 1989) were shown to retain the binding characteristics of the parent antibody. However, this is not achievable on a routine basis: most naturally occurring antibodies need both a V_(H) and a V_(L) to retain full immunoreactivity. For example, in the case of V_(H) D1.3 (Ward et al., 1989), although it still binds hen egg lysozyme (HEL) with an affinity close to that of the parent antibody, it was shown that loss of specificity was observed in that it can no longer distinguish turkey lysozyme from HEL, whereas the Fv can (Berry and Davies, 1992). Although murine dABs can be obtained more routinely from spleen libraries (Ward et al., 1989), the approach is unsustainable because of the many problems associated with their production and physical behaviour: expression is extremely poor, affinity tends to be low, stability and solubility in water is low, and non-specific binding is usually very high. According to the literature a possible explanation of these undesirable characteristics is the exposure of the hydrophobic residues which are normally buried in the V_(H) -V_(L) interface. The exposed hydrophobic patches are thought to contribute to aggregation of the protein inside the cells and/or in the culture medium, leading to poor expression and/or poor solubility (Anthony et al., 1992; Ward et al., 1989). The hydrophobic patches can also explain the high non-specific binding described by Berry and Davies, 1992. These problems clearly limit the usefulness of these molecules. Most of the Camelid antibodies appear to be an exception to this rule in that they only need one V-domain, namely V_(H), to specifically and effectively bind an antigen (Hamers-Castermans et al., 1993). In addition, preliminary data indicate that they seem not to suffer from the disadvantages of mouse dABs, as these camelid antibodies or fragments thereof are soluble and have been shown to express well in yeast and Aspergillus moulds. These observations can have important consequences for the production and exploitation of antibody-based products, see patent application WO 94/25591 (UNILEVER et al., first priority date Apr. 29, 1993).

2. Production of Antibody Fragments

Several microbial expression systems have already been developed for producing active antibody fragments, e.g. the production of Fab in various hosts, such as E. coli (Better et al., 1988, Skerra and Pluckthun, 1988, Carter et al., 1992), yeast (Horwitz et al., 1988), and the filamentous fungus Trichoderma reesei (Nyyssonen et al., 1993) has been described. The recombinant protein yields in these alternative systems can be relatively high (1-2 g/l for Fab secreted to the periplasmic space of E. coli in high cell density fermentation, see Carter et al., 1992), or at a lower level, e.g. about 0.1 mg/l for Fab in yeast in fermenters (Horwitz et al., 1988), and 150 mg/l for a fusion protein CBHI-Fab and 1 mg/l for Fab in Trichoderma in fermenters (Nyyssonen et al., 1993) and such production is very cheap compared to whole antibody production in mammalian cells (hybridoma, myeloma, CHO). Although the latter can give yields of the order of 1 g/l in high cell density fermentation, it is a time-consuming and very expensive manufacturing method resulting in a cost price of about 1000 .English Pound./gram of antibody. It was further demonstrated that plants can be used as hosts for the production of both whole antibodies (Hiatt et al., 1989) and scFv's (Owen et al., 1992, Firek et al., 1993), whereby yields of up to 0.5% of the total soluble protein content in tobacco leaves were mentioned.

The fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a V_(H) and a V_(L) can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv (Bird et al. (1988), Huston et al. (1988), and granted patent EP-B-0281604 (GENEX/ENZON LABS INC.; first priority date Sep. 2, 1986).

3. Bivalent and Bispecific Antibodies and Antibody Fragments

The antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site. Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the V_(H) of an Fv (Cumber et al., 1992), or at the C-terminus of the V_(L) of an scFv (Pack and Pluckthun, 1992), or through the hinge cysteine residues of Fab's (Carter et al., 1992). Another approach to produce bivalent antibody fragments is described by Kostelny et al. (1992) and Pack and Pluckthun (1992) and is based on the inclusion of a C-terminal peptide that promotes dimerization.

When two different specificities are desired, one can generate bispecific antibody fragments. The traditional approach to generate bispecific whole antibodies was to fuse two hybridoma cell lines each producing an antibody having the desired specificity. Because of the random association of immunoglobulin heavy and light chains, these hybrid hybridomas produce a mixture of up to 10 different heavy and light chain combinations, only one of which is the bispecific antibody (Milstein and Cuello, 1983). Therefore, these bispecific antibodies have to be purified with cumbersome procedures, which considerably decrease the yield of the desired product.

Alternative approaches include in-vitro linking of two antigen specificities by chemical cross-linking of cysteine residues either in the hinge or via a genetically introduced C-terminal Cys as described above. An improvement of such in vitro assembly was achieved by using recombinant fusions of Fab's with peptides that promote formation of heterodimers (Kostelny et al., 1992). However, the yield of bispecific product in these methods is far less than 100%.

A more efficient approach to produce bivalent or bispecific antibody fragments, not involving in vitro chemical assembly steps, was described by Holliger et al. (1993). This approach takes advantage of the observation that scFv's secreted from bacteria are often present as both monomers and dimers. This observation suggested that the V_(H) and V_(L) of different chains can pair, thus forming dimers and larger complexes. The dimeric antibody fragments, also named "diabodies" by Hollinger et al., in fact are small bivalent antibody fragments that assembled in vivo. By linking the V_(H) and V_(L) of two different antibodies 1 and 2, to form "cross-over" chains V_(H) 1V_(L) 2 and V_(H) 2-V_(L) 1 (see FIG. 2B), the dimerisation process was shown to reassemble both antigen-binding sites. The affinity of the two binding sites was shown to be equal to the starting scFv's, or even to be 10-fold increased when the polypeptide linker covalently linking V_(H) and V_(L) was removed, thus generating two proteins each consisting of a V_(H) directly and covalently linked to a V_(L) not pairing with the V_(H) (see FIG. 2C). This strategy of producing bispecific antibody fragments was also described in several patent applications. Patent application WO 94/09131 (SCOTGEN LTD; priority date Oct. 15, 1992) relates to a bispecific binding protein in which the binding domains are derived from both a V_(H) and a V_(L) region either present at two chains or linked in an scFv, whereas other fused antibody domains, e.g. C-terminal constant domains, are used to stabilise the dimeric constructs. Patent application WO 94/13804 (CAMBRIDGE ANTIBODY TECHNOLOGY/MEDICAL RESEARCH COUNCIL; first priority date Dec. 4, 1992) relates to a polypeptide containing a V_(H) and a V_(L) which are incapable of associating with each other, whereby the V-domains can be connected with or without a linker.

Mallender and Voss, 1994 (also described in patent application WO 94/13806; DOW CHEMICAL CO; priority date Dec. 11, 1992) reported the in vivo production of a single-chain bispecific antibody fragment in E. coli. The bispecificity of the bivalent protein was based on two previously produced monovalent scFv molecules possessing distinct specificities, being linked together at the genetic level by a flexible polypeptide linker. The thus formed V_(H) 1-linker-V_(L) 1-linker-V_(H) 2-linker-V_(L) 2 fragment (see FIG. 2A) was shown to contain both antigen binding specificities 1 and 2. (1=anti-fluorescein, 2=anti-single-stranded DNA). Traditionally, whenever single-chain antibody fragments are referred to, a single molecule consisting of one heavy chain linked to one (corresponding) light chain in the presence or absence of a polypeptide linker is implicated. When making bivalent or bispecific antibody fragments through the `diabody` approach (Holliger et al., (1993) and patent application WO 94/09131) or by the `double scFv` approach (Mallender and Voss, 1994 and patent application WO 94/13806), again the V_(H) is linked to a (the corresponding) V_(L).

It is realised that claims 32 and 33 of patent application WO 93/11161 (ENZON INC.; priority date Nov. 25, 1991) and the corresponding passages in that specification on page 22, lines 1-10 may read on a polypeptide comprising two V_(L) 's fused together via a flexible polypeptide linker, and on a polypeptide comprising two V_(H) 's fused together via a flexible polypeptide linker, respectively. However, no examples were given to substantiate this approach, thus it was in fact a hypothetical possibility instead of an actually produced compound.

A skilled person would not have expected that such approach would be viable for at least three reasons. Firstly, it is widely recognised that immunoglobulin heavy chains (excluding the above described camel immunoglobulins) have very limited solubility and spontaneously precipitate out of aqueous solution when isolated from their light chain partners. Secondly, several groups have shown (Ward et al., 1989, Berry and Davies, 1992, and Anthony et al., 1992) that expression of V_(H) 's in the absence of V_(L) 's is hampered by extremely poor yields of unstable product with many undesirable properties, e.g. non-specific binding. Thirdly in patent application WO 94/13804 it was described on page 31 lines 10-12, that in computer modelling experiments they could not model as heterodimers V_(H) --V_(H) and V_(L) --V_(L) given the constraints of short linkers.

Thus the simple suggestion given in patent application WO 93/11161 is not an enabling disclosure leading a skilled person to try with a reasonable expectation of success whether such suggestion would work; therefore, that patent application should not be considered as relevant prior art for the present invention.

SUMMARY OF THE INVENTION

The present invention provides a bispecific or bivalent antibody fragment analogue, which comprises a binding complex containing two polypeptide chains, one of which comprises two times a variable domain of a heavy chain (V_(H)) in series and the other comprises two times a variable domain of a light chain (V_(L)) in series.

In one aspect of the invention one chain of the antibody fragment analogue comprises a first V_(H) (V_(H) -A) connected to a second V_(H) (V_(H) -B) and the other chain comprises a first V_(L) (V_(L) -A) connected to a second V_(L) (V_(L) -B). In a preferred embodiment of this aspect one chain comprises a first V_(H) (V_(H) -A) followed by a second V_(H) (V_(H) -B), thus [V_(H) -A * V_(H) -B], and the other chain comprises a first V_(L) (V_(L) -A) preceded by a second V_(L) (V_(L) -B), thus [V_(L) -B * V_(L) -A]. For some embodiments of this aspect the two V_(H) 's are directly connected to each other, but for other embodiments of this aspect of the invention the two V_(L) 's are directly connected to each other. According to another embodiment of this aspect of the invention the two V_(H) 's are connected to each other by a linker and also the two V_(L) 's are connected to each other by a linker. Such a linker usually comprises at least one amino acid residue.

According to a special embodiment of this aspect of the invention one chain comprises a first V_(H) (V_(H) -A) followed by a second V_(H) (V_(H) -B), thus [V_(H) -A * V_(H) -B], and the other chain comprises a first V_(L) (V_(L) -A) followed by a second V_(L) (V_(L) -B), thus [V_(L) -A * V_(L) -B], and in which the two V_(H) 's are connected to each other by a linker and also the two V_(L) 's are connected to each other by a linker, whereas each linker comprises at least 10 amino acid residues.

According to the above aspect of the invention with A being different from B there are provided bispecific antibody fragment analogues.

According to another aspect of the invention the specificities A and B are the same resulting in a bivalent antibody fragment.

According to a further aspect of the invention the bispecific or bivalent antibody fragment analogues can be used in a diagnostic technique or for immunoassays, in a purification method, for therapy, or in other methods in which immunoglobulins or fragments thereof are used. Such uses are well-known in the art.

The invention also provides a process for producing the antibody fragments of the invention in that a host is transformed by incorporating into that host a DNA encoding the two V_(H) 's with or without a connecting linker and a DNA encoding the two V_(L) 's with or without a connecting linker. Preferably the two DNA's are placed in a dicistronic arrangement.

It is also possible that the two linked V_(H) 's and the two linked V_(L) 's are produced separately by different hosts, after which the linked V_(H) 's produced by one host can be combined with the linked V_(L) 's produced by the other host. The hosts can be selected from the group consisting of prokaryotic bacteria of which examples are Gram-negative bacteria, e.g. E. coli, and Gram-positive bacteria, e.g. B. subtilis or lactic acid bacteria, lower eukaryotes examples of which are yeasts, e.g. belonging to the genera Saccharomyces, Kluyveromyces, or Trichoderma, moulds, e.g. belonging to the genera Aspergillus and Neurospora, and higher eukaryotes, examples of which are plants, e.g. tobacco, and animal cells, examples of which are myeloma cells and CHO cells. The techniques to transform a host by genetic engineering methods in order to have a desirable polypeptide produced by such host are well-known to persons skilled in the art as is evident from the literature mentioned above under the heading "Background of the invention and prior art".

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts in schematic form the structure of a typical antibody (immunoglobulin) molecule.

FIG. 2 shows a schematic representation of published arrangements of heavy and light chain V-domain gene fragments that have been proven to produce bispecific antibody fragments.

FIG. 3 shows in diagrammatic form the suggested arrangement of the V-domains of a double head antibody fragment according to the invention with the V-domains in the following order: V_(H) A-V_(H) B+V_(L) B-V_(L) A.

FIG. 4 shows the nucleotide sequence of the EcoRI-HindIII insert of pUR.4124 containing DNA (see SEQ ID NO: 1) encoding V_(L) Lys-Linker-V_(H) Lys (see SEQ ID NO: 2).

FIG. 5 shows the nucleotide sequence of the HindIII-EcoRI insert of plasmid Fv.3418 (see SEQ ID NO: 3) containing DNA encoding pelB leader-V_(H) 3418 (see SEQ ID NO: 4) and DNA encoding pelB leader-V_(L) 3418 (see SEQ ID NO: 5).

FIG. 6 shows the nucleotide sequence of the HindIII-EcoRI insert of plasmid Fv.4715-myc (see SEQ ID NO: 6) containing DNA encoding pelB leader-V_(H) 4715 (see SEQ ID NO: 7) and DNA encoding pelB leader-V_(L) 4715-Myc tag (see SEQ ID NO: 8).

FIG. 7 shows the nucleotide sequence of the HindIII-EcoRI insert of scFv.4715-myc containing DNA (see SEQ ID NO: 9) encoding pelB leader-V_(H) 4715-Linker-V_(L) 4715-Myc tag (see SEQ ID NO: 10).

FIGS. 8A and 8B show the nucleotide sequence of the HindIII-EcoRI insert of pGOSA.E (see SEQ ID NO: 11) containing DNA encoding pelB leader-V_(H) 4715-Linker-V_(L) 3418 (see SEQ ID NO: 12) and DNA encoding pelB leader-V_(L) 3418-Linker-V_(H) 4715 (see SEQ ID NO: 13).

FIG. 9 gives an overview of the oligonucleotides and their positions in pGOSA.E that can be used to replace V-domain gene fragments.

FIG. 10 illustrates the amino acid sequence of the V_(H--V) _(H) and V_(L) --V_(L) domain junctions in fusion polypeptides GOSA.E (see amino acids 114-145 in SEQ ID NO: 12 and amino acids 102-128 in SEQ ID NO: 13), GOSA.V (see SEQ ID NO: 30 and amino acids 102-128 in SEQ ID NO: 13), GOSA.S (see amino acids 114-145 in SEQ ID NO: 12 and SEQ ID NO: 31) and GOSA.T (see SEQ ID NO: 30 and SEQ ID NO: 31).

FIG. 11 shows the specificity of Streptococcus binding of scFv.4715-myc.

FIG. 12 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.E.

FIG. 13 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.V.

FIG. 14 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.S.

FIG. 15 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.T.

FIG. 16 shows the results of an ELISA. Individual fractions of a gelfiltration experiment using partially purified GOSA.E as feedstock were tested for glucose oxidase and Streptococcus sanguis bispecific binding activity.

FIG. 17 shows the results of an ELISA. Individual fractions of a gelfiltration experiment using partially purified GOSA.V as feedstock were tested for glucose oxidase and Streptococcus sanguis bispecific binding activity.

FIG. 18 shows the results of an ELISA. Individual fractions of a gelfiltration experiment using partially purified GOSA.T as feedstock were tested for glucose oxidase and Streptococcus sanguis bispecific binding activity.

FIG. 19 shows the source of fragment PCR.I BstEII/SacI.

FIG. 20 shows the source of fragment PCR.II SfiI/EcoRI.

FIG. 21 shows the source of fragment PCR.III NheI/SacI.

FIG. 22 shows the source of fragment PCR.IV XhoI/EcoRI.

FIG. 23 shows the source of fragment PCR.V SalI/EcoRI.

FIG. 24 shows the source of fragment PCR.VI SfiI/NheI.

FIG. 25 shows the source of fragment PCR.VII BstEII/NheI.

FIG. 26 shows the source of fragment PCR.VIII XhoI/EcoRI.

FIG. 27 shows the source of fragment PCR.IX BstEII/NheI.

FIG. 28 shows the source of fragment PCR.X PstI/EcoRI.

FIG. 29 shows the construction of plasmid pGOSA.A.

FIG. 30 shows the construction of plasmid pGOSA.B.

FIG. 31 shows the construction of plasmid PGOSA.C.

FIG. 32 shows the construction of plasmid pGOSA.D.

FIG. 33 shows the construction of plasmid pGOSA.E.

FIG. 34 shows the construction of plasmid pGOSA.V.

FIG. 35 shows the construction of plasmid pGOSA.S.

FIG. 36 shows the construction of plasmid PGOSA.T.

FIGS. 37A and 37B show the construction of plasmid pGOSA.G.

FIG. 38 shows the construction of plasmid pGOSA.J.

FIG. 39 shows the construction of plasmid pGOSA.Z.

FIG. 40 shows the construction of plasmid pGOSA.AA.

FIG. 41 shows the construction of plasmid pGOSA.AB.

FIG. 42 shows the construction of plasmid PGOSA.L.

FIG. 43 shows the construction of plasmid PGOSA.Y.

FIG. 44 shows the construction of plasmid pGOSA.X.

FIG. 45 shows the construction of plasmid pGOSA.AC.

FIG. 46 shows the construction of plasmid pGOSA.AD.

Table 1 shows the nucleotide sequence of the oligonucleotides used to produce the constructs described in this specification. Restriction sites encoded by these primers are underlined.

Table 2 gives an overview of all GOSA constructs described in this specification.

Table 2A describes intermediate constructs that were not further tested.

Table 2B describes the dicistronic constructs.

Table 2C describes the monocistronic constructs.

DETAILED DESCRIPTION OF THE INVENTION

In this specification the construction of an antibody fragment analogue consisting of a two chain protein complex is described, in which one of the chains consists of two heavy chain V-domains and the other chain consists of the two corresponding light chain V-domains in either order. The variable domains are linked either directly or through a polypeptide linker. Subsequent molecular modelling of this combination suggested that the protein chains could fold such that both binding sites are fully accessible, provided that the connecting linkers are kept long enough to span 30 to 35 Å.

Whereas in patent application WO 93/11161 it is explicitly described that for the above described bispecific complexes two flexible polypeptide linkers in the self assembling complex are required, the present invention illustrated here describes in particular the construction of a two chain protein complex containing only one linker or no linkers at all. The latter antibody fragment analogue thus consists of a two chain protein complex containing one polypeptide chain comprising heavy chain V-domains fused directly together and another polypeptide chain comprising the corresponding light chain V-domains fused together, both fusions in the absence of linkers. But also two chain protein complexes in which each chain comprises a linker between the two variable domains can be used as antibody fragment analogues according to the invention as described below with construct pGOSA.E. However, the two chain complexes containing only one linker or no linker at all are preferred. The abbreviation GOSA used in this specification relates to a combination of glucose oxidase and Streptococcus sanguis.

In this specification evidence is provided that these antibody fragment analogues ("double heads") contain both antigen binding specificities of the Fv's used to generate these bispecific antibody fragments. It is exemplified that these type of constructs according to the invention can be used to target the enzyme glucose oxidase to whole bacteria, using antibody fragments derived from hybridomas expressing antibodies directed against these antigens.

The present invention is now described by reference to some specific examples, which are included for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLES General Experimental Strains, Plasmids and Media

All cloning steps were performed in E. coli JM109 (endA1, recA1, gyrA96, thi, hsdR17(r_(K), m_(K) ⁺), relA1, supE44, Δ(lac-proAB), [F', traD36, proAB, lacI^(q) ZΔM15]. E. coli cultures were grown in 2×TY medium (16 g tryptone, 10 g yeast extract, 5 g NaCl per liter H₂ O), where indicated supplemented with 2% glucose and/or 100 μg/ml ampicillin. Transformations were plated out on SOBAG plates (20 g tryptone, 5 g yeast extract, 15 g agar, 0.5 g NaCl per liter H₂ O plus 10 mM MgCl₂, 2% glucose, 100 μg/ml ampicillin) The expression vectors used are derivatives of pUC19. The oligonucleotide primers used in the PCR reactions were synthesized on an Applied Biosystems 381A DNA Synthesiser by the phosphoramidite method.

Expression of GOSA Constructs

Colonies from freshly transformed JM109 plated onto SOBAG plates were used to inoculate 2×TY medium supplemented with 100 μg/ml ampicillin, 2% glucose. Cultures were shaken at 37° C. to an OD₆₀₀ in the range of 0.5 to 1.0. Cells were pelleted by centrifugation and the supernatant was removed. The pelleted cells were resuspended in 2×TY medium with 100 μg/ml ampicillin, 1 mM IPTG, and grown for a further 18 hours at 25° C. Cells were pelleted by centrifugation and the supernatant, containing the secreted chains, used directly in an ELISA. The proteins in the periplasm of the pelleted cells were extracted by resuspending the cell pellet in 1/20 of the original culture volume of lysis buffer (20% sucrose, 200 mM Tris-HCl pH 7.5, 1 mM EDTA, 500 μg/ml lysozyme). After incubation at 25° C. for 20 minutes an equal volume of H₂ O was added and the incubation was continued for another 20 minutes. The suspension was spun at 10.000 g for 15 minutes and the supernatant containing the periplasmic proteins was used directly in an ELISA.

ELISA

96 well ELISA plates (Greiner HC plates) were activated overnight at 37° C. with 200 μl/well of an 1/10 dilution of an over night culture of Streptococcus cells in 0.05 M sodium carbonate buffer at pH=9.5. Following one wash with PBST, the antigen sensitised plates were pre-blocked for 1 hour at 37° C. with 200 μl/well blocking buffer (2% BSA, 0.15% Tween in PBS). Samples containing 50 μl blocking buffer plus 50 μl culture supernatants or periplasmic cell extracts (neat or diluted with PBS) were added to the Streptococcus sensitised plate and incubated for 2 hours at 37° C. Following 4 washes with PBS-T, 100 μl of blocking buffer containing glucose oxidase (50 μg/ml) was added to every well. After incubation at 37° C. for 1 hour unbound glucose oxidase was removed by 4 washes with PBS-T. Bound glucose oxidase was detected by adding 100 μl substrate to each well (70 mM Na-citrate, 320 mM Na-phosphate, 27 mg/ml glucose, 0.5 μg/ml HRP, 100 μg/ml TMB). The colour reaction was stopped after 1 hour by the addition of 35 μl 2 M HCl and the A450 was measured (compare FIGS. 11/15).

Affinity Purification of GOSA Antibody Fragments

GOSA.E, GOSA.V, GOSA.S and GOSA.T were partially purified by affinity chromatography. 100 ml periplasmic extract of each of these constructs was loaded onto a Glucose-oxidase-Sepharose column (CNBr-Sepharose, Pharmacia) prepared according to the manufacturer's instructions. After extensive washes with PBS the bound GOSA antibody fragments were eluted in 0.1M glycine buffer at pH=2.8. The fractions were neutralised with Tris and analysed by polyacrylamide gel electrophoresis followed by silver staining and tested for the presence of double head activity.

EXAMPLE 1 Construction of the pGOSA Double Head Expression Vectors

In this Example the construction of a two chain protein complex is described, in which one of the chains consists of two heavy chain V-domains and the other chain consists of the two corresponding light chain V-domains. The variable domains are linked either directly or through a polypeptide linker. The expression vectors used are derivatives of a pUC19 derived plasmid containing a HindIII-EcoRI fragment that in the case of plasmid scFv.4715-myc contains a DNA fragment encoding one pelB signal sequence fused to the N-terminus of the V_(H) that is directly linked to the corresponding V_(L) of the antibody through a connecting flexible peptide linker, (Gly₄ Ser)₃ (present in SEQ ID NO: 2 as amino acids 109-123 and in SEQ ID NO: 10 as amino acids 121-135), thus generating a single-chain molecule (see FIG. 7).

In the dual-chain Fv and the pGOSA expression vectors, the DNA fragments encoding both the V_(H) and V_(L) of the antibody are preceded by a ribosome binding site and a DNA sequence encoding the pelB signal sequence in an artificial dicistronic operon under the control of a single inducible promoter (see FIGS. 5, 6 and 8A and 8B). Expression of these constructs is driven by the inducible lacZ promoter. The nucleotide sequence of the HindIII-EcoRI inserts of the plasmids pUR.4124, Fv.3418, Fv.4715-myc and scFv.4715-myc constructs used for the generation of the bispecific antibody fragments are given in FIGS. 4-7, respectively. Moreover, a culture of E. coli cells harbouring plasmid scFv.4715-myc and a culture of E. coli cells harbouring plasmid Fv.3418 were deposited under the Budapest Treaty at the National Collection of Type Cultures (Central Public Health Laboratory) in London (United Kingdom) with deposition numbers NCTC 12916 and NCTC 12915, respectively. In agreement with Rule 28 (4) EPC, or a similar arrangement for a State not being a Contracting State of the EPC, it is hereby requested that a sample of such deposit, when requested, will be submitted to an expert only. The construction of PGOSA.E (see FIGS. 8A and 8B for the HindIll-EcoRI insert of pUC19) involved several cloning steps. The appropriate restriction sites in the various domains were introduced by PCR directed mutagenesis using the oligonucleotides listed in Table 1 below. The PGOSA.E derivatives pGOSA.V, pGOSA.S and PGOSA.T with only one or no linker sequence are derived from the pGOSA.E construct by removing the linker sequences by means of PCR directed mutagenesis with oligonucleotides listed in Table 1 below.

                                      TABLE 1                                      __________________________________________________________________________     DBL.1                                                                               5'-CAC CAT CTC CAG AGA CAA TGG CAA G-3'                                                                       (= SEQ ID NO: 14)                          DBL.2                                                                               5'-GAG CGC GAG CTC GGC CGA ACC GGC C.sup.1 GA TCC GCC                                                         (= SEQ ID NO: 15)                               ACC GCC AGA GCC-3'                                                        DBL.3                                                                               5'-CAG GAT CCG GCC GGT TCG GCC.sup.1  CAG GTC CAG CTG                                                         (= SEQ ID NO: 16)                               CAA CAG TCA GGA-3'                                                        DBL.4                                                                               5'-CTA CAT GAA TTC.sup.2  GCT AGC.sup.3  TTA TTA TGA GGA GAC                                                  (= SEQ ID NO: 17)                               GGT GAC GGT GGT CCC TTG GC-3'                                             DBL.5                                                                               5'-TAA TAA GCT AGC.sup.3  GGA GCT GCA TGC AAA TTC TAT                                                         (= SEQ ID NO: 18)                               TTC-3'                                                                    DBL.6                                                                               5'-ACC AAG CTC GAG.sup.4  ATC AAA CGG GG-3'                                                                   (= SEQ ID NO: 19)                          DBL.7                                                                               5'-AAT GTC GAA TTC.sup.2  GTC GAC.sup.5  TCC GCC ACC GCC AGA                                                  (= SEQ ID NO: 20)                               GCC-3'                                                                    DBL.8                                                                               5'-ATT GGA GTC GAC.sup.5  ATC GAA CTC ACT CAG TCT CCA                                                         (= SEQ ID NO: 21)                               TTC TCC-3'                                                                DBL.9                                                                               5'-TGA AGT GAA TTC.sup.2  GCG GCC GC.sup.6 T TAT TAC CGT TTG                                                  (= SEQ ID NO: 22)                               ATT TCG AGC TTG GTC CC-3'                                                 DBL.10                                                                              5'-CGA ATT CGG TCA CC.sup.8 G TCT CCT CAC AGG TCC AGT                                                         (= SEQ ID NO: 23)                               TGC AAC AG-3'                                                             DBL.11                                                                              5'-CGA ATT CTC GAG.sup.4  ATC AAA CGG GAC ATC GAA CTC                                                         (= SEQ ID NO: 24)                               ACT CAG TCT CC-3'                                                         DBL.12                                                                              5'-CGA ATT CGG TCA CC.sup.8 G TCT CCT CAC AGG TGC AGT                                                         (= SEQ ID NO: 25)                               TGC AGG AG-3'                                                             PCR.51                                                                              5'-AGG T(C/G)(A/C) A(C/A)C TGC AG.sup.7 (C/G) AGT C(A/T)G                                                     (= SEQ ID NO: 26)                               G-3'                                                                      PCR.89                                                                              5'-TGA GGA GAC GGT GAC C.sup.8 GT GGT CCC TTG GCC CC-3'                                                       (= SEQ ID NO: 27)                          PCR.90                                                                              5'-GAC ATT GAG CTC.sup.9  ACC CAG TCT CCA-3'                                                                  (= SEQ ID NO: 28)                          PCR.116                                                                             5'-GTT AGA TCT CGA G.sup.4 CT TGG TCC C-3'                                                                    (= SEQ ID NO: 29)                          __________________________________________________________________________      1=SfiI, 2=EcoRI, 3=NheI, 4=XhoI, 5=SalI, 6=NotI, 7=PstI, 8=BstEII, 9=SacI

These three constructs lack some of the restriction sites at the new joining points. The V_(H) A-V_(H) B gene fragment without a linker lacks the 5' V_(H) B SfiI site. The V_(L) B-V_(L) A gene fragment without a linker lacks the 5' V_(L) A SalI site. The position of the oligonucleotides in the PGOSA constructs given in Table 1 are shown in FIG. 9. The pGOSA expression vectors and the oligonucleotides in Table 1 have been designed to enable most specificities to be cloned into the pGOSA constructs. FIG. 10 shows the amino acid sequence of the junctions between the V_(H) A-V_(H) B and V_(L) B-V_(L) A fragments encoded by DNA present in pGOSA.E, pGOSA.V, pGOSA.S and pGOSA.T. A more detailed description of the preparation of pGOSA.E, pGOSA.V, PGOSA.S and pGOSA.T is given in Example 5.

EXAMPLE 2 Bifunctional Binding Activity of GOSA Double Heads

In this Example we provide evidence that the above described molecules ("double heads"), i.e. the two chain protein complexes, contain both antigen binding specificities of the Fv's used to generate these multi-functional antibody fragment analogues. FIG. 12-15 show that GOSA.E, GOSA.V, GOSA.S and GOSA.T can be used to specifically target the enzyme glucose oxidase to several Streptococcus sanguis strains using antibody fragments derived from hybridoma's expressing antibodies directed against these antigens.

Comparison of the binding specificity of the GOSA constructs (see FIGS. 12-15) and the binding specificity of the scFv.4715-myc (see FIG. 11) shows that the fine specificity of the anti-Streptococcus sanguis scFv.4715 is preserved in the GOSA "double heads".

EXAMPLE 3 FPLC Analysis of GOSA Double Heads

Partially purified GOSA.E, GOSA.V, GOSA.S and GOSA.T samples (estimated to be 50-80% pure by polyacrylamide gel electrophoresis) were analysed on a Pharmacia FPLC Superose 12 column. The analysis was performed using PBS at a flow rate of 0.3 ml/minute. Eluate was monitored at 280 nm and 0.3 ml fractions were collected and analysed by ELISA. Usually GOSA.E, GOSA.V, GOSA.S and GOSA.T samples only gave one GOSA double head activity peak as determined by ELISA (see FIGS. 16-18). The position of this peak in the elution pattern indicated that the molecular weight of the GOSA double head is 40-50 kD. Since this molecular weight corresponds to the expected molecular weight of the V_(H) 2+V_(L) 2 double head dimer, the conclusion is justified that GOSA.E, GOSA.V, GOSA.S and GOSA.T are primarily produced as dimeric molecules. Occasionally an activity peak with an apparent molecular weight of ≈200 kD was observed (see FIG. 16). The presence of Glucose Oxidase activity in these fractions (data not shown) indicate that these fractions contain GOSA double head complexed with glucose oxidase that was eluted with the GOSA sample from the glucose oxidase-sepharose affinity matrix.

EXAMPLE 4 Production of Other Double Heads

The methods described in the previous Examples were used to produce other double heads, which also appeared to be active against the antigens for which they were developed. These other double heads had the following specificities:

anti-S. sanguis/anti-beta-HCG,

anti-S. sanguis/anti-urease,

anti-S. sanguis/anti-hen-egg-lysozyme,

anti-beta-HCG/anti-hen-egg-lysozyme,

anti-hen-egg-lysozyme/anti-glucose oxidase,

anti-huIgG/anti-glucose oxidase,

anti-urease/anti-glucose oxidase,

anti-lacto-peroxidase/anti-glucose oxidase,

anti-alpha-HCG/anti-glucose oxidase, and

anti-reactive-Red-6/anti-glucose oxidase.

EXAMPLE 5 Detailed Description of the Preparation of Intermediate Constructs pGOSA.A, pGOSA.B pGOSA.C and pGOSA.D and Their Use for the Preparation of Plasmid pGOSA.E and Its Derivatives PGOSA.V, PGOSA.S and PGOSA.T Oligonucleotides and PCR

The primary structures of the oligonucleotide primers used in the construction of the bispecific `pGOSA` constructs are shown in Table 1 above. Reaction mixture used for amplification of DNA fragments were 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl₂, 50 mM KCl, 0.01% gelatin (w/v), 0.1% Triton X-100, 400 mM of each dNTP, 5.0 units of Vent DNA polymerase (New England Biolabs), 100 ng of template DNA, and 500 ng of each primer (for 100 μl reactions). Reaction conditions were: 94° C. for 4 minutes, followed by 33 cycles of each 1 minute at 94° C., 1 minute at 55° C., and 1 minute 72° C.

Plasmid DNA\Vector\Insert Preparation and Ligation\Transformation

Plasmid DNA was prepared using the `Qiagen P-100 Midi-DNA Preparation` system. Vectors and inserts were prepared by digestion of 10 μg (for vector preparation) or 20 μg (for insert preparation) with the specified restriction endonucleases under appropriate conditions (buffers and temperatures as specified by suppliers). Modification of the DNA ends with Klenow DNA polymerase and dephosphorylation with Calf Intestine Phosphorylase were performed according to the manufacturers instructions. Vector DNAs and inserts were separated through agarose gel electrophoresis and purified with DEAE-membranes NA45 (Schleicher & Schuell) as described by Maniatis et al. Ligations were performed in 20 μl volumes containing 30 mM Tris-HCl pH 7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 300-400 ng vector DNA, 100-200 ng insert DNA and 1 Weiss unit T₄ DNA ligase. After ligation for 2-4 h at room temperature, CaCl₂ competent E. coli JM109 (Maniatis) were transformed using 7.5 μl ligation reaction. The transformation mixtures were plated onto SOBAG plates and grown overnight at 37° C. Correct clones were identified by restriction analysis and verified by automated dideoxy sequencing (Applied Biosystems).

Restriction Digestion of PCR Products

Following amplification each reaction was checked for the presence of a band of the appropriate size by agarose gel electrophoresis. One or two 100 μl PCR reaction mixtures of each of the PCR reactions PCR.I-PCR.X (FIG. 20-29), together containing approximately 2-4 μg DNA product were subjected to phenol-chloroform extraction, chloroform extraction and ethanol precipitation. The DNA pellets were washed twice with 70% ethanol and allowed to dry. Next, the PCR products were digested overnight (18 h) in the presence of excess restriction enzyme in the following mixes at the specified temperatures and volumes.

PCR.I

50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 50 mM NaCl, 4 mM spermidine, 0.4 μg/ml BSA, 4 μl (=40 U) SacI, 4 μl (=40 U) BstEII, in 100 μl total volume at 37° C.

PCR.II

10 mM Tris-Acetate pH 7.5, 10 mM MgAc₂, 50 mM KAc (1x "One-Phor-All" buffer ex Pharmacia), 4 μl (=48 U) SfiI, in 50 μl total volume at 50° C. under mineral oil. After overnight digestion, PCR.II-SfiI was digested with EcoRI (overnight at 37° C.) by the addition of 16 μl H₂ O, 30 μl 10x "One-Phor-All" buffer (Phanmacia) (100 mM Tris-Acetate pH 7.5, 100 mM MgAc₂, 500 mM KAc) and 4 μl (=40 U) EcoRI.

PCR. III

10 mM Tris-Acetate pH 7.5, 10 mM MgAc₂, 50 mM KAc (1x "One-Phor-All" buffer {Pharmacia}), 4 μl (=40 U) NheI, 4 μl (=40 U) SacI, in 100 μl total volume at 37° C.

PCR.IV

20 mM Tris-Acetate pH 7.5, 20 mM MgAc₂, 100 mM KAc (2x "One-Phor-All" buffer {Pharmacia}), 4μl (=40 U) XhoI, 4 μl (=40 U) EcoRI, in 100 μl total volume at 37° C.

PCR.V

20 nM Tris-Acetate pH 7.5, 20 mM MgAc₂, 100 mM KAc (2x "One-Phor-All" buffer {Pharmacia}), 4 μl (=40 U) SalI, 4 μl (=40 U) EcoRI, in 100 μl total volume at 37° C.

PCR.VI

10 mM Tris-Acetate pH 7.5, 10 mM MgAc₂, 50 mM KAc (1x "One-Phor-All" buffer {Pharmacia}), 4 μl (=48 U) SfiI, in 50 μl total volume at 50° C. under mineral oil. After overnight digestion, PCR.VI-SfiI was digested with NheI (overnight at 37° C.) by the addition of 41 μl H₂ O, 5 μl 10x "One-Phor-All" buffer (Pharmacia) (100 mM Tris-Acetate pH 7.5, 100 mM MgAc₂, 500 mM KAc) and 4 μl (=40 U) NheI.

PCR.VII

50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 50 mM NaCl, 4 mM spermidine, 0.4 μg/ml BSA, 4 μl (=40 U) NheI, 4 μl (=40 U) BstEII, in 100 μl total volume at 37° C.

PCR.VIII

20 mM Tris-Acetate pH 7.5, 20 mM MgAc₂, 100 mM KAc (2x "One-Phor-All" buffer {Pharmacia}), 4 μl (=40 U) EcoRI, in 50 μl total volume at 37° C. After overnight digestion, PCR.VIII-EcoRI was digested with XhoI (overnight at 37° ) by the addition of 46 μl H₂ O and 4 μl (=40 U) XhoI.

PCR.IX

25 mM Tris-Acetate, pH 7.8, 100 mM KAc, 10 mM MgAc, 1 mM DTT (1x "Multi-Core" buffer {Promega}), 4 mM spermidine, 0.4 μg/ml BSA, 4 μl (=40 U) NheI, 4 μl (=40 U) BstEII, in 100 μl total volume at 37° C.

PCR.X

50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 50 mM NaCl, 4 mM permidine, 0.4 μg/ml BSA, 4 μl (=40 U) PstI, 4 μl (=40 U) EcoRI, in 100 μl total volume at 37° C.

    ______________________________________                                         The digested PCR fragments                                                     ______________________________________                                         PCR.I-SacI/BstEII,  PCR.II-SfiI/EcoRI,                                         PCR.III-NheI/SacI,  PCR.IV-XhoI/EcoRI,                                         PCR.V-SalI/EcoRI,   PCR.VI-SfiI/NheI,                                          PCR.VII-BstEII/NheI,                                                                               PCR.VIII-XhoI/EcoRI,                                       PCR.IX-BstEII/NheI, and                                                                            PCR.X-PstI/EcoRI                                           ______________________________________                                    

were purified on an 1.2% agarose gel using DEAE-membranes NA45 (Schleicher & Schuell) as described by Maniatis et al. The purified fragments were dissolved in H₂ O at a concentration of 100-150 ng/μl.

Construction of the pGOSA Double Head Expression Vectors

The construction of pGOSA.E (see FIGS. 8A and 8B) involved several cloning steps that produced 4 intermediate constructs PGOSA.A to pGOSA.D (see FIG. 29-33). The final expression vector pGOSA.E and the oligonucleotides in Table 1 above have been designed to enable most specificities to be cloned into the final pGOSA.E construct (FIG. 9). The upstream V_(H) domain can be replaced by any PstI-BstEII V_(H) gene fragment obtained with oligonucleotides PCR.51 and PCR.89 (see Table 1 above). The oligonucleotides DBL.3 and DBL.4 (see Table 1 above) were designed to introduce SfiI and NheI restriction sites in the V_(H) gene fragments thus allowing cloning of those V_(H) gene fragments into the Sfil-NheI sites as the downstream V_(H) domain. All V_(L) gene fragments obtained with oligonucleotides PCR.116 and PCR.90 (see Table 1 above) can be cloned into the position of the V_(L).3418 gene fragment as a SacI-XhoI fragment. A complication here however is the presence of an internal SacI site in the V_(H).3418 gene fragment. Oligonucleotides DBL.8 and DBL.9 (see Table 1 above) are designed to allow cloning of V_(L) gene fragments into the position of the V_(L).4715 gene fragment-as a SalI-NotI fragment. The pGOSA.E derivatives PGOSA.V, pGOSA.S and PGOSA.T with only one or no linker sequences contain some aberrant restriction sites at the new joining points. The V_(H) A-V_(H) B construct without a linker lacks the 5' V_(H) B SfiI site. The V_(H) B fragment is cloned into these constructs as a BstEII/NheI fragment using oligonucleotides DBL.10 or DBL.11 and DBL.4 (see Table 1 above). The V_(L) B-V_(L) A construct without a linker lacks the 5' V_(L) A SalI site. The V_(L) A fragment is cloned into these constructs as a XhoI/EcoRI fragment using oligonucleotides DBL.11 and DBL.9 (see Table 1 above).

In the following part of the description the following linkers are mentioned which are also present in the sequence listing:

the (Gly₄ Ser)₃ linker, present in SEQ ID NO: 2 as amino acids 109-123 and SEQ ID NO: 10 as amino acids 121-135,

the (Gly₄ Ser)₃ AlaGlySerAla linker (=linkerA), present in SEQ ID NO: 12 as amino acids 121-139, and

the (Gly₄ Ser)₂ Gly₄ Val linker (=linkerV), present in SEQ ID NO: 13 as amino acids 108-122.

pGOSA.A

This plasmid is derived from both the Fv.4715-myc construct and the scFv.4715-myc construct.

An SfiI restriction site was introduced between the DNA sequence encoding the (Gly₄ Ser)₃ linker and the gene fragment encoding the V_(L) of the scFv.4715-myc construct (see FIG. 30). This was achieved by replacing the BstEII-SacI fragment of the latter construct by the fragment PCR-I BstEII/SacI (FIG. 19) that contains an SfiI site between the DNA encoding the (Gly₄ Ser)₃ linker and the V_(L).4715 gene fragment. The introduction of the SfiI site also introduced 4 additional amino acids (AlaGlySerAla) between the (Gly₄ Ser)₃ linker and V_(L).4715 resulting in a (Gly₄ Ser)₃ AlaGlySerAla linker (linkerA). The oligonucleotides used to produce PCR-I (DBL.1 and DBL.2, see Table 1 above) were designed to match the sequence of the framework-3 region of V_(H).4715 and to prime at the junction of the DNA encoding the (Gly₄ Ser)₃ linker and the V_(L).4715 gene fragment, respectively. Thus PGOSA.A can be indicated as: pelB-V_(H) 4715-linkerA- (SfiI) -V_(L) 4715-myc.

pGOSA.B

This plasmid is derived from plasmid Fv.3418 (see FIG. 31). The XhoI-EcoRI fragment of plasmid Fv.3418 comprising the 3' end of DNA encoding framework-4 of the V_(L) including the stop codon was removed and replaced by the fragment PCR-IV XhoI/EcoRI (FIG. 22). The oligonucleotides used to produce PCR-IV (DBL.6 and DBL.7, see Table 1 above) were designed to match the sequence at the junction of the V_(L) and the (Gly₄ Ser)₃ linker perfectly (DBL.6), and to be able to prime at the junction of the (Gly₄ Ser)₃ linker and the V_(H) in pUR.4124 (DBL.7). DBL.7 removed the PstI site in the V_(H) (silent mutation) and introduced a SalI restriction site at the junction of the (Gly₄ Ser)₃ linker and the V_(H), thereby replacing the last Ser of the linker by a Val residue resulting in a (Gly₄ Ser)₂ Gly₄ Val linker (linkerV). Thus pGOSA.B can be indicated as:

pelB-V_(H) 3418+pelB-V_(L) 3418-linkerV- (SalI-EcoRI)

pGOSA.C

This plasmid contains DNA encoding V_(H).4715 linked by the (Gly₄ Ser)₃ AlaGlySerAla linker to V_(H).3418 (see FIG. 31), thus: pelB-V_(H) 4715-linkerA-V_(H) 3418.

This construct was obtained by replacing the SfiI-EcoRI fragment from pGOSA.A encoding V_(L).4715 by the fragment PCR-II SfiI/EcoRI containing the V_(H).3418 gene (see FIG. 20). The oligonucleotides used to produce PCR-II (DBL.3 and DBL.4, see Table 1 above) hybridize in the framework-1 and framework-4 region of the gene encoding V_(H).3418, respectively. DBL.3 was designed to remove the PstI restriction site (silent mutation) and to introduce an SfiI restriction site upstream of the V_(H) gene. DBL.4 destroys the BstEII restriction site in the framework-4 region and introduces an NheI restriction site downstream of the stopcodon.

pGOSA.D

This plasmid contains a dicistronic operon comprising the V_(H).3418 gene and DNA encoding V_(L).3418 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to V_(L).4715 (see FIG. 32), thus:

pelB-V_(H) 3418+pelB-V_(L) 3418-linkerV-V_(L) 4715.

This construct was obtained by digesting plasmid pGOSA.B with SalI-EcoRI and inserting the fragment PCR-V SalI/EcoRI (FIG. 23) containing the V_(L).4715 gene. The oligonucleotides used to obtain PCR-V (DBL.8 and DBL.9, see Table 1 above) were designed to match the nucleotide sequence of the framework-1 and framework-4 regions of the V_(L).4715 gene, respectively. DBL.8 removed the SacI site from the framework-1 region (silent mutation) and introduced a SalI restriction site upstream of the V_(L).4715 gene. DBL.9 destroyed the XhoI restriction site in the framework-4 region of the V_(L).4715 gene (silent mutation) and introduced a NotI and an EcoRI restriction site downstream of the stop codon.

PGOSA.E

This plasmid contains a dicistronic operon comprising DNA encoding V_(H).4715 linked by the (Gly4Ser)₃ AlaGlySerAla linker to V_(H).3418 plus DNA encoding V_(L).3418 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to V_(L).4715 (see FIG. 33), thus:

pelB-V_(H) 4715-linkerA-V_(H) 3418+pelB-V_(L) 3418-linkerV-V_(L) 4715.

Both translational units are preceded by a ribosome binding site and DNA encoding a pelB leader sequence. This plasmid was obtained by a three-point ligation by mixing the vector resulting from pGOSA.D after removal of the V_(H) 3418-encoding PstI-SacI insert with the PstI-NheI pGOSA.C insert containing V_(H).4715 linked to V_(H).3418 and the PCR-III NheI/SacI fragment (see FIG. 21). The remaining PstI-SacI pGOSA.D vector contains the 5' end of the framework-1 region of V_(H) 3418 up to the PstI restriction site and V_(L).3418 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to V_(L).4715 starting from the SacI restriction site in V_(L).3418. The PstI-NheI PGOSA.C insert contains V_(H).4715 linked by the (Gly₄ Ser)₃ -AlaGlySerAla linker to V_(H).3418, starting from the PstI restriction site in the framework-1 region in V_(H).4715. The NheI-SacI PCR-III fragment provides the ribosome binding site and DNA encoding the pelB leader sequence for the V_(L).3418-(Gly₄ Ser)₂ Gly₄ Val-V_(L).4715 construct. The oligo-nucleotides DBL.5 and PCR.116 (see Table 1 above) used to generate PCR-III were designed to match the sequence upstream of the ribosome binding site of V_(L).4715 in Fv.4715 and to introduce an NheI restriction site (DBL.5), and to match the framework-4 region of V_(L).3418 (PCR.116).

pGOSA.V

This plasmid is derived from PGOSA.E (see FIG. 35) from which the BstEII/NheI fragment containing DNA encoding linkerA-V_(H).3418 was excised and replaced by the fragment PCR-VII BstEII/NheI containing the V_(H).3418 gene (see FIG. 25). The resulting plasmid pGOSA.V contains V_(H).3418 linked directly to the framework-4 region of V_(H).4715, plus V_(L).4715 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to the framework-4 region of V_(L).3418, thus:

pelB-V_(H) 4715*V_(H) 3418+pelB-V_(L) 3418-linkerV-V_(L) 4715.

pGOSA.S

This plasmid is derived from pGOSA.E (see FIG. 35) from which the (Gly₄ Ser)₂ Gly₄ Val-V_(L) 4715 XhoI/EcoRI fragment was excised and replaced by the fragment PCR-VIII XhoI/EcoRI which contains V_(L).4715 (see FIG. 26). The resulting plasmid pGOSA.S contains V_(H).4715 linked by the (Gly₄ Ser)₃ -AlaGlySerAla linker to V_(H).3418 plus V_(L).3418 linked directly to the 5' end of the framework-1 region of V_(L).4715, thus:

pelB-V_(H).4715-linkerA-V_(H).3418+pelB-V_(L).3418*V_(L).4715.

pGOSA.T

This plasmid contains a dicistronic operon consisting of V_(H).3418 directly to the framework-4 region of V_(H).4715 plus V_(L).3418 linked directly to the 5' end of the framework-1 region of V_(L).4715 (see FIG. 36). Both transcriptional units are preceded by a ribosome binding site and a pelB leader sequence, thus:

pelB-V_(H).4715*V_(H).3418+pelB-V_(L).3418*V_(L).4715.

This construct was obtained by inserting the NheI/EcoRI fragment of pGOSA.S which contains V_(L).3418 linked directly to the 5' end of the framework-1 region of V_(L).4715, into the vector pGOSA.V from which the NheI/EcoRI fragment containing V_(L).3418 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to V_(L).4715 was removed.

EXAMPLE 6 Detailed Description of the Preparation of Other Dicistronic Constructs pGOSA.G, and pGOSA.J, pGOSA.Z, PGOSA.AA and PGOSA.AB pGOSA.G

This plasmid is an intermediate for the synthesis of pGOSA.J. It is derived from pGOSA.E from which the V_(H) 4715 PstI/BstEII fragment has been excised and replaced by the V_(H) 3418 PstI/BstEII fragment (excised from Fv.3418). The resulting plasmid pGOSA.G (see FIGS. 37A and 37B) contains two copies of V_(H).3418 linked by the (Gly₄ Ser)₃ AlaGlySerAla linker, plus V_(L).4715 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to the framework-4 region of V_(L).3418, thus:

pelB-V_(H).3418-linkerA-V_(H).3418+pelB-V_(L).3418-linkerV-V_(L).47

pGOSA.J

This plasmid contains a dicistronic operon consisting of V_(H).3418 linked by the (Gly₄ Ser)₃ AlaGlySerAla linker to V_(H).4715 plus V_(L).3418 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to V_(L).4715. Both transcriptional units are preceded by a ribosome binding site-and a pelB leader sequence (see FIG. 39), thus:

pelB-V_(H).3418-linkerA-V_(H).4715+pelB-V_(L).3418-linkerV-V_(L).47

This construct was obtained by inserting the fragment PCR-VI SfiI/NheI which contains V_(H) 4715 (FIG. 24), into the vector pGOSA.G from which the SfiI/NheI V_(H) 3418 fragment was removed.

pGOSA.Z

This plasmid is derived from PGOSA.G from which the (Gly₄ Ser)₃ AlaGlySerAla linker-V_(H) 3418 BstEII/NheI fragment was excised and replaced by the fragment PCR-IX BstEII/NheI which contains V_(H).4715 (FIG. 27). The resulting plasmid pGOSA.Z (see FIG. 39) contains V_(H).3418 linked directly to the framework-1 region of V_(H).4715, plus V_(L).4715 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to the framework-4 region of V_(L).3418, thus:

pelB-V_(H).3418*V_(H).4715+pelB-V_(L).3418-linkerV-V_(L).4715

PGOSA.AA

This plasmid contains a dicistronic operon consisting of the V_(H).3418 linked directly to the 5' end of the framework-1 region of V_(H).4715 plus V_(L).3418 linked directly to the 5' end of the framework-1 region of V_(L).4715. Both transcriptional units are preceded by a ribosome binding site and a pelB leader sequence (see FIG. 40). This construct was obtained by inserting the NheI/EcoRI fragment of PGOSA.T which contains V_(L).3418 linked directly to the 5' end of the framework-1 region of V_(L).4715, into the vector pGOSA.Z from which the NheI/EcoRI fragment containing V_(L).3418 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to V_(L).4715 was removed, thus:

pelB-V_(H).3418*V_(H).4715+pelB-V_(L).3418*V_(L).4715

pGOSA.AB

This plasmid is derived from pGOSA.J by a three point ligation reaction (see FIG. 41). The SacI/EcoRI insert, containing part of V_(H).3418 and the full (Gly4Ser)₃ AlaGlySerAla linker-V_(H).4715 and the V_(L).3418-(Gly₄ Ser)₂ Gly₄ Val-V_(L).4715 encoding sequences, was removed and replaced by the SacI/SacI pGOSA.J fragment containing the same part of V_(H).3418 and the full (Gly₄ Ser)₃ AlaGlySerAla linker-V_(H).4715 and the SacI/EcoRI PGOSA.T fragment containing V_(L).3418 linked directly to the framework-1 region of V_(L).4715 (see FIG. 36). The resulting plasmid contains V_(H).3418 linked by the (Gly₄ Ser)₃ AlaGlySerAla linker to the 5' end of the framework-1 region of V_(H).4715 plus V_(L).3418 linked directly to the 5' end of the framework-1 region of V_(L).4715, thus:

pelB-V_(H).3418-linkerA-V_(H).4715+pelB-V_(L).3418*V_(L).4715

EXAMPLE 7 Detailed Description of the Preparation of Monocistronic Constructs pGOSA.L and pGOSA.Y, and pGOSA.C, pGOSA.X. pGOSA.AC and pGOSA.AD pGOSA.L

This plasmid is derived from pGOSA.E from which the HindlIl/NheI fragment containing DNA encoding V_(H).4715-(Gly₄ Ser)₃ AlaGlySerAla-V_(H).3418 was removed (see FIG. 42). The DNA ends of the vector were made blunt-end using Klenow DNA polymerase and ligated. The resulting plasmid pGOSA.L contains V_(L).3418 linked by the (Gly₄ Ser)₂ Gly₄ Val linker to the 5' end of the framework-1 region of V_(L).4715, thus:

pelB-V_(L).3418-linkerV-V_(L).4715.

pGOSA.Y

This plasmid is derived from pGOSA.T from which the HindIII/NheI fragment containing DNA encoding V_(H).4715-V_(H).3418 was removed (see FIG. 43). The DNA ends of the vector were made blunt-end using Klenow DNA polymerase and ligated. The resulting plasmid pGOSA.Y contains V_(L).3418 linked directly to 5' end of the framework-1 region of V_(L).4715, thus:

pelB-V_(L).3418*V_(L).4715.

The preparation of pGOSA.C was given in Example 5 above; it can be indicated with: pelB-V_(H).4715-linkerA-V_(H).3418.

pGOSA.X

This plasmid is derived from pGOSA.T from which the RheI/EcoRI fragment containing DNA encoding V_(L).3418-V_(L).4715 was removed. The DNA ends of the vector were made blunt-end using Klenow DNA polymerase and ligated. The resulting plasmid pGOSA.X (see FIG. 44) contains V_(H).4715 linked directly to 5' end of the framework-1 region of V_(H).3418, thus: pelB-V_(H).4715*V_(H).3418.

pGOSA.AC

This plasmid is derived from pGOSA.Z from which the NheI/EcoRI fragment containing DNA encoding V_(L).3418-(Gly₄ Ser)₂ Gly₄ Val-V_(L).4715 was removed (see FIG. 45). The DNA ends of the vector were made blunt-end using Klenow DNA polymerase and ligated. The resulting plasmid pGOSA.AC contains V_(H).3418 linked directly to 5' end of the framework-1 region of V_(H).4715, thus:

pelB-V_(H).3418*V_(H).4715.

pGOSA.AD

This plasmid was obtained by inserting the PstI/EcoRI PCR.X. fragment containing DNA encoding V_(H).3418- (Gly₄ Ser)₃ AlaGly-SerAla-V_(H).4715 (see FIG. 28) into the Fv.4715-myc vector from which the PstI/EcoRI Fv.4715-myc insert was removed (see FIG. 46), thus: pelB-V_(H).3418-linkerA-V_(H).4715.

These monocistronic constructs can be used to transform the same host with two different plasmids or to transform two different hosts, so that the two V_(H) 's in series can be produced separately from the two V_(L) 's in series.

Evaluation of the Results Obtained

Bifunctional binding activity of GOSA double heads

In this specification the construction of a two chain protein complex is described, in which one of the chains consists of two heavy chain V-domains and the other chain consists of the two corresponding light chain V-domains. The variable domains are linked either directly or through a polypeptide linker. In this specification evidence is provided that these type of molecules ("double heads") contain both antigen binding specificities of the Fv's used to generate these multi-functional antibody fragments. FIG. 12 shows that GOSA.E can be used to specifically target the enzyme glucose oxidase to several Streptococcus sanguis strains, using antibody fragments derived from hybridomas expressing antibodies directed against these antigens. FIG. 12 further shows that the fine specificity of the anti-Streptococcus sanguis scFv 4715 is preserved in the GOSA.E double head.

Effect of Linkers and Relative Position of V-domains on Double Head Activity

After it was shown that the "cross-over double-head" approach (V_(H) A-V_(H) B+V_(L) B-V_(L) A) yields active bispecific molecules, the importance of the relative position of the V-domains in these constructs was investigated. Both possible positional orientations (GOSA.E=V_(H) A-LinkerA-V_(H) B+V_(L) B-LinkerV-V_(L) A and GOSA.J=V_(H) B-LinkerA-V_(H) A+V_(L) B-LinkerV-V_(L) A) were constructed and tested for bispecific activity, despite the suggestion obtained by molecular modelling that the binding site formed by the second (downstream/C-terminal) V-domains in the configuration V_(H) B-V_(H) A+V_(L) B-V_(L) A (GOSA.J) was in an unfavourable position for binding to large protein antigens on the surface of cells. Surprisingly however, it was found experimentally that the downstream binding site is in fact accessible. Although the relative position of the heavy chains and the light chains was found to have an effect on the observed reactivity both tested combinations show bispecific activity with the "cross-over" combination (GOSA.E=V_(H) A-V_(H) B+V_(L) B-V_(L) A) exhibiting a higher level of reactivity compared to the combination V_(H) B-V_(L) A+V_(L) B-V_(L) (=GOSA.J) as demonstrated for A=anti-Strep and B=anti-Gox.

Molecular modelling of the V_(H) B-V_(H) A+V_(L) B-V_(L) A (=GOSA.J) configuration further suggested that, only when the connecting linkers are kept long enough (to span 30 to 35 Å), the protein chains could fold such that both binding sites are fully accessible.

The "cross-over" configuration: V_(H) A-V_(H) B+V_(L) B-V_(L) A (GOSA.E) wherein linker length was not critical, was predicted to result in a complex with both binding sites facing in opposite directions, without the restraints suggested for the configuration V_(H) B-V_(H) A+V_(L) B-V_(L) A (GOSA.J). Removing the flexible polypeptide linker from the V_(H) A-V_(H) B chain only had a minimal effect on the ability of the double head in the "cross-over" configuration (GOSA.V=V_(H) A*V_(H) B+V_(L) B-V_(L) A) to bind both S. sanguis and Glucose oxidase. However, removing the flexible polypeptide linker from the V_(H) B-V_(H) A chain from the molecule in the V_(H) B-V_(H) A+V_(L) B-V_(L) A configuration (GOSA.Z =V_(H) B*V_(H) A+V_(L) B-V_(L) A) resulted in a dramatic reduction of its ability to bind both S. sanguis and Glucose oxidase.

In contrast with the double head in the "cross-over" configuration without the flexible polypeptide linker between the two heavy chain domains (GOSA.V), where molecular modelling predicted the resulting molecule to be active, removal of the flexible linker from the V_(L) B-V_(L) A chain could not be modelled such that both binding sites were fully accessible. ELISA results confirm that the double head in the V_(H) B-V_(H) A+V_(L) B-V_(L) A configuration without a linker between the two light chain domains (GOSA.AB) exhibits only minimal S. sanguis and glucose oxidase binding activity. Surprisingly, deletion of the flexible linker from the V_(L) B-V_(L) A chain from the double head in the "cross-over" configuration (GOSA.S) only had a small effect on the bispecific activity of the resulting molecule. As expected from the molecular modelling results from the double heads without a flexible linker between the two light chain domains, removal of both the flexible polypeptide linkers from the double head molecules, could not be modelled such that both binding sites were fully accessible. In agreement with the ELISA results obtained with the GOSA.AB construct, the double head in the V_(H) B-V_(H) A+V_(L) B-V_(L) A configuration without any linkers (GOSA.AA) only exhibits minimal if any S. sanguis and glucose oxidase binding activity. Surprisingly, the double head in the "cross-over" configuration without any linkers (GOSA.T=V_(H) A*V_(H) B+V_(L) B*V_(L) A) still exhibited 25-50% of S. sanguis and glucose oxidase bispecific binding activity when compared to the activity of the double head in the "cross-over" configuration with two linkers (GOSA.E).

Thus the conclusion of this work is that modelling can give some indications, but that the computer programmes cannot predict what is possible and what not. Several deviations from the modelling expectations were found. With a paraphrase on an old saying: theories are nice but the experiment is the ultimate proof.

Sensitivity of GOSA Double Heads

Using an ELISA format it was shown that the sensitivity of the GOSA.E double head is as least as a sensitive as an IgG-glucose oxidase conjugate, as determined by the lowest concentration of Streptococcus sanguis antigen immobilised on a solid phase that is still detectable.

GOSA Double Heads Are Produced as Dimers

FPLC analysis of partially affinity-purified GOSA.E, GOSA.V, GOSA.S and GOSA.T samples usually gave only one GOSA double head activity peak as determined by ELISA (FIGS. 16-18). The position of this peak in the elution pattern indicated that the molecular weight of the GOSA double head is 40-50 kD. Since this molecular weight corresponds to the expected molecular weight of the V_(H) 2+V_(L) 2 double head dimer, it was concluded that GOSA.E, GOSA.V, GOSA.S and GOSA.T are primarily produced as dimeric molecules. Occasionally an activity peak with an apparent molecular weight of ≈200 kD was observed (FIG. 16). The presence of glucose oxidase activity in these fractions indicate that these fractions contain GOSA double head complexed with glucose oxidase.

In Vitro Assembly of GOSA Double Heads

It was shown that bifunctionally active dimeric GOSA molecules together in one cell can be produced by translation from one dicistronic messenger (GOSA.E, GOSA.S, GOSA.T, GOSA.V, GOSA.J, GOSA.AB, GOSA.AA and GOSA.Z). In addition high levels of S. sanguis and glucose oxidase bispecific binding activity is formed when supernatants of cultures producing the separate GOSA subunits are mixed (see Example 7). The effects of linkers and the relative position of the individual V_(H) -domains on the S. sanquis and glucose oxidase bispecific binding activity observed in these mixing experiments are comparable to the dicistronic constructs.

The constructs described above are summarised in Table 2 below.

Table 2A describes intermediate constructs that were not further tested.

Table 2B describes the dicistronic constructs.

Table 2C describes the monocistronic constructs.

(LiA) stands for the V_(H) --V_(H) linker (Gly₄ Ser)₃ AlaGlySerAla (=linkerA)

(LiV) stands for the V_(L) --V_(L) linker (Gly₄ Ser)₂ Gly₄ Val (=linkerV)

                  TABLE 2                                                          ______________________________________                                         Table 2A                                                                       GOSA.A:   V.sub.H.4715-LiA-(SfiI)-V.sub.L.4715-myc                             GOSA.B:   V.sub.H.3418-LiV-V.sub.L.3418-(SalI/EcoRI)                           GOSA.D:   V.sub.H.3418 + V.sub.L.3418-LiV-V.sub.L.4715                         GOSA.G:   V.sub.H.3418-LiA-V.sub.H.3418 + V.sub.L.3418-LiV-V.sub.L.4715        Table 2B                                                                       GOSA.E:   V.sub.H.4715-LiA-V.sub.H.3418 + V.sub.L.3418-LiV-V.sub.L.4715        GOSA.S:   V.sub.H.4715-LiA-V.sub.H.3418 + V.sub.L.3418*V.sub.L.4715            GOSA.T:   V.sub.H.4715*V.sub.H.3418 + V.sub.L.3418*V.sub.L.4715                GOSA.V:   V.sub.H.4715*V.sub.H.3418 + V.sub.L.3418-LiV-V.sub.L.4715            GOSA.J:   V.sub.H.3418-LiA-V.sub.H.4715 + V.sub.L.3418-LiV-V.sub.L.4715        GOSA.AB:  V.sub.H.3418-LiA-V.sub.H.4715 + V.sub.L.3418*V.sub.L.4715            GOSA.AA:  V.sub.H.3418*V.sub.H.4715 + V.sub.L.3418*V.sub.L.4715                GOSA.Z:   V.sub.H.3418*V.sub.H.4715 + V.sub.L.3418-LiV-V.sub.L.4715            Table 2C                                                                       GOSA.L:   V.sub.L.3418-LiV-V.sub.L.4715                                        GOSA.Y:   V.sub.L.3418*V.sub.L.4715                                            GOSA.AD:  V.sub.H.3418-LiA-V.sub.H.4715                                        GOSA.AC:  V.sub.H.3418*V.sub.H.4715                                            GOSA.C:   V.sub.H.4715-LiA-V.sub.H.3418                                        GOSA.X:   V.sub.H.4715*V.sub.H.3418                                            ______________________________________                                          (*) indicates that the two heavy chain domains or the two light chain          domains are fused together without a connecting linker.                  

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    __________________________________________________________________________     #             SEQUENCE LISTING                                                 - (1) GENERAL INFORMATION:                                                     -    (iii) NUMBER OF SEQUENCES: 31                                             - (2) INFORMATION FOR SEQ ID NO: 1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 737 base                                                           (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "cDNA domains with syntheticc                                                              linker(s)"                                                      -    (vii) IMMEDIATE SOURCE:                                                   #insert of pUR4124E: EcoRI-HindIII                                             -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:11..730                                                           (D) OTHER INFORMATION:/pro - #duct= "VLlys-GS-VHlys"                 -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:11..334                                                           (D) OTHER INFORMATION:/pro - #duct= "VLlys"                          -     (ix) FEATURE:                                                                      (A) NAME/KEY: misc.sub.-- - #RNA                                               (B) LOCATION:335..379                                                          (D) OTHER INFORMATION:/pro - #duct= "(Gly4Ser)3 linker"              -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:380..727                                                          (D) OTHER INFORMATION:/pro - #duct= "VHlys"                          #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - GAATTCGGCC GAC ATC GAG CTC ACC CAG TCT CCA GC - #C TCC CTT TCT GCG             49                                                                           #Thr Gln Ser Pro Ala Ser Leu Ser Ala                                           #         10                                                                   - TCT GTG GGA GAA ACT GTC ACC ATC ACA TGT CG - #A GCA AGT GGG AAT ATT            97                                                                           Ser Val Gly Glu Thr Val Thr Ile Thr Cys Ar - #g Ala Ser Gly Asn Ile            #    25                                                                        - CAC AAT TAT TTA GCA TGG TAT CAG CAG AAA CA - #G GGA AAA TCT CCT CAG           145                                                                           His Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gl - #n Gly Lys Ser Pro Gln            #45                                                                            - CTC CTG GTC TAT TAT ACA ACA ACC TTA GCA GA - #T GGT GTG CCA TCA AGG           193                                                                           Leu Leu Val Tyr Tyr Thr Thr Thr Leu Ala As - #p Gly Val Pro Ser Arg            #                60                                                            - TTC AGT GGC AGT GGA TCA GGA ACA CAA TAT TC - #T CTC AAG ATC AAC AGC           241                                                                           Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Se - #r Leu Lys Ile Asn Ser            #            75                                                                - CTG CAA CCT GAA GAT TTT GGG AGT TAT TAC TG - #T CAA CAT TTT TGG AGT           289                                                                           Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cy - #s Gln His Phe Trp Ser            #        90                                                                    - ACT CCT CGG ACG TTC GGT GGA GGG ACC AAG CT - #C GAG ATC AAA CGG GGT           337                                                                           Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Le - #u Glu Ile Lys Arg Gly            #    105                                                                       - GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GG - #T GGC GGA TCG CAG GTG           385                                                                           Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gl - #y Gly Gly Ser Gln Val            110                 1 - #15                 1 - #20                 1 -        #25                                                                            - CAG CTG CAG GAG TCA GGA CCT GGC CTG GTG GC - #G CCC TCA CAG AGC CTG           433                                                                           Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Al - #a Pro Ser Gln Ser Leu            #               140                                                            - TCC ATC ACA TGC ACC GTC TCA GGG TTC TCA TT - #A ACC GGC TAT GGT GTA           481                                                                           Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Le - #u Thr Gly Tyr Gly Val            #           155                                                                - AAC TGG GTT CGC CAG CCT CCA GGA AAG GGT CT - #G GAG TGG CTG GGA ATG           529                                                                           Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Le - #u Glu Trp Leu Gly Met            #       170                                                                    - ATT TGG GGT GAT GGA AAC ACA GAC TAT AAT TC - #A GCT CTC AAA TCC AGA           577                                                                           Ile Trp Gly Asp Gly Asn Thr Asp Tyr Asn Se - #r Ala Leu Lys Ser Arg            #   185                                                                        - CTG AGC ATC AGC AAG GAC AAC TCC AAG AGC CA - #A GTT TTC TTA AAA ATG           625                                                                           Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gl - #n Val Phe Leu Lys Met            190                 1 - #95                 2 - #00                 2 -        #05                                                                            - AAC AGT CTG CAC ACT GAT GAC ACA GCC AGG TA - #C TAC TGT GCC AGA GAG           673                                                                           Asn Ser Leu His Thr Asp Asp Thr Ala Arg Ty - #r Tyr Cys Ala Arg Glu            #               220                                                            - AGA GAT TAT AGG CTT GAC TAC TGG GGC CAA GG - #G ACC ACG GTC ACC GTC           721                                                                           Arg Asp Tyr Arg Leu Asp Tyr Trp Gly Gln Gl - #y Thr Thr Val Thr Val            #           235                                                                #   737            TT                                                          Ser Ser                                                                        - (2) INFORMATION FOR SEQ ID NO: 2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 239 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Le - #u Ser Ala Ser Val Gly          #                15                                                            - Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gl - #y Asn Ile His Asn Tyr          #            30                                                                - Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Se - #r Pro Gln Leu Leu Val          #        45                                                                    - Tyr Tyr Thr Thr Thr Leu Ala Asp Gly Val Pr - #o Ser Arg Phe Ser Gly          #    60                                                                        - Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Il - #e Asn Ser Leu Gln Pro          #80                                                                            - Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Ph - #e Trp Ser Thr Pro Arg          #                95                                                            - Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ly - #s Arg Gly Gly Gly Gly          #           110                                                                - Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gln Val Gln Leu Gln          #       125                                                                    - Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gl - #n Ser Leu Ser Ile Thr          #   140                                                                        - Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Ty - #r Gly Val Asn Trp Val          145                 1 - #50                 1 - #55                 1 -        #60                                                                            - Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Le - #u Gly Met Ile Trp Gly          #               175                                                            - Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Ly - #s Ser Arg Leu Ser Ile          #           190                                                                - Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Le - #u Lys Met Asn Ser Leu          #       205                                                                    - His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Al - #a Arg Glu Arg Asp Tyr          #   220                                                                        - Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Va - #l Thr Val Ser Ser              225                 2 - #30                 2 - #35                            - (2) INFORMATION FOR SEQ ID NO: 3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 920 base                                                           (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "cDNA domains with syntheticc                                                              linker(s)"                                                      -    (vii) IMMEDIATE SOURCE:                                                   #insert Fv.3418LONE: HindIII-EcoRI                                             -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:36..443                                                           (D) OTHER INFORMATION:/pro - #duct= "pelB-VH3418"                    -     (ix) FEATURE:                                                                      (A) NAME/KEY: sig.sub.-- - #peptide                                            (B) LOCATION:36..101                                                           (D) OTHER INFORMATION:/pro - #duct= "pectate lyase"                  -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:102..440                                                          (D) OTHER INFORMATION:/pro - #duct= "VH3418"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:495..884                                                          (D) OTHER INFORMATION:/pro - #duct= "pelB-VL4318"                    -     (ix) FEATURE:                                                                      (A) NAME/KEY: sig.sub.-- - #peptide                                            (B) LOCATION:495..560                                                #/product= "pectate lyase"ATION:                                               -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:561..881                                                          (D) OTHER INFORMATION:/pro - #duct= "VL3418"                         #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #CTA TTG CCT        53C AAGGAGACAG TCATA ATG AAA TAC                           #   Met Lys Tyr Leu Leu Pro                                                    20                                                                             - ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GC - #C CAA CCA GCG ATG GCC           101                                                                           Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Al - #a Gln Pro Ala Met Ala            - CAG GTG CAG CTG CAG CAG TCA GGA CCT GAG CT - #G GTA AAG CCT GGG GCT           149                                                                           Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Le - #u Val Lys Pro Gly Ala            #                15                                                            - TCA GTG AAG ATG TCC TGC AAG GCT TCT GGA TA - #C ACA TTC ACT AGC TAT           197                                                                           Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ty - #r Thr Phe Thr Ser Tyr            #            30                                                                - GTT ATG CAC TGG GTG AAA CAG AAG CCT GGG CA - #G GGC CTT GAG TGG ATT           245                                                                           Val Met His Trp Val Lys Gln Lys Pro Gly Gl - #n Gly Leu Glu Trp Ile            #        45                                                                    - GGA TAT ATT TAT CCT TAC AAT GAT GGT ACT AA - #G TAC AAT GAG AAG TTC           293                                                                           Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Ly - #s Tyr Asn Glu Lys Phe            #    60                                                                        - AAA GGC AAG GCC ACA CTG ACT TCA GAC AAA TC - #C TCC AGC ACA GCC TAC           341                                                                           Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Se - #r Ser Ser Thr Ala Tyr            #80                                                                            - ATG GAG CTC AGC AGC CTG ACC TCT GAG GAC TC - #T GCG GTC TAT TAC TGT           389                                                                           Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Se - #r Ala Val Tyr Tyr Cys            #                95                                                            - TCA AGA CGC TTT GAC TAC TGG GGC CAA GGG AC - #C ACG GTC ACC GTC TCC           437                                                                           Ser Arg Arg Phe Asp Tyr Trp Gly Gln Gly Th - #r Thr Val Thr Val Ser            #           110                                                                - TCA TAA TAAGAGCTAT GGGAGCTTGC ATGCAAATTC TATTTCAAGG AG - #ACAGTCAT            493                                                                           Ser                                                                            #GGA TTG TTA TTA CTC        539 GCA GCC GCT                                      Met Lys Tyr Leu Leu Pro Thr Ala Ala A - #la Gly Leu Leu Leu Leu              10                                                                             - GCT GCC CAA CCA GCG ATG GCC GAC ATC GAG CT - #C ACC CAG TCT CCA TCT           587                                                                           Ala Ala Gln Pro Ala Met Ala Asp Ile Glu Le - #u Thr Gln Ser Pro Ser            #     5  1                                                                     - TCC ATG TAT GCA TCT CTA GGA GAG AGA ATC AC - #T ATC ACT TGC AAG GCG           635                                                                           Ser Met Tyr Ala Ser Leu Gly Glu Arg Ile Th - #r Ile Thr Cys Lys Ala            #25                                                                            - AGT CAG GAC ATT AAT ACC TAT TTA ACC TGG TT - #C CAG CAG AAA CCA GGG           683                                                                           Ser Gln Asp Ile Asn Thr Tyr Leu Thr Trp Ph - #e Gln Gln Lys Pro Gly            #                40                                                            - AAA TCT CCC AAG ACC CTG ATC TAT CGT GCA AA - #C AGA TTG CTA GAT GGG           731                                                                           Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala As - #n Arg Leu Leu Asp Gly            #            55                                                                - GTC CCA TCA AGG TTC AGT GGC AGT GGA TCT GG - #G CAA GAT TAT TCT CTC           779                                                                           Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gl - #y Gln Asp Tyr Ser Leu            #        70                                                                    - ACC ATC AGC AGC CTG GAC TAT GAA GAT ATG GG - #A ATT TAT TAT TGT CTA           827                                                                           Thr Ile Ser Ser Leu Asp Tyr Glu Asp Met Gl - #y Ile Tyr Tyr Cys Leu            #    85                                                                        - CAA TAT GAT GAG TTG TAC ACG TTC GGA GGG GG - #G ACC AAG CTC GAG ATC           875                                                                           Gln Tyr Asp Glu Leu Tyr Thr Phe Gly Gly Gl - #y Thr Lys Leu Glu Ile            #105                                                                           #                 920CAA ACGGTATAAG GATCCAGCTC GAATTC                          Lys Arg                                                                        - (2) INFORMATION FOR SEQ ID NO: 4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 135 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl - #y Leu Leu Leu Leu Ala          10                                                                             - Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gl - #n Gln Ser Gly Pro Glu          #  10                                                                          - Leu Val Lys Pro Gly Ala Ser Val Lys Met Se - #r Cys Lys Ala Ser Gly          #                25                                                            - Tyr Thr Phe Thr Ser Tyr Val Met His Trp Va - #l Lys Gln Lys Pro Gly          #            40                                                                - Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pr - #o Tyr Asn Asp Gly Thr          #        55                                                                    - Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Th - #r Leu Thr Ser Asp Lys          #    70                                                                        - Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Se - #r Leu Thr Ser Glu Asp          #90                                                                            - Ser Ala Val Tyr Tyr Cys Ser Arg Arg Phe As - #p Tyr Trp Gly Gln Gly          #                105                                                           - Thr Thr Val Thr Val Ser Ser                                                              110                                                                - (2) INFORMATION FOR SEQ ID NO: 5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 129 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl - #y Leu Leu Leu Leu Ala          10                                                                             - Ala Gln Pro Ala Met Ala Asp Ile Glu Leu Th - #r Gln Ser Pro Ser Ser          #  10                                                                          - Met Tyr Ala Ser Leu Gly Glu Arg Ile Thr Il - #e Thr Cys Lys Ala Ser          #                25                                                            - Gln Asp Ile Asn Thr Tyr Leu Thr Trp Phe Gl - #n Gln Lys Pro Gly Lys          #            40                                                                - Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Ar - #g Leu Leu Asp Gly Val          #        55                                                                    - Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gl - #n Asp Tyr Ser Leu Thr          #    70                                                                        - Ile Ser Ser Leu Asp Tyr Glu Asp Met Gly Il - #e Tyr Tyr Cys Leu Gln          #90                                                                            - Tyr Asp Glu Leu Tyr Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys          #                105                                                           - Arg                                                                          - (2) INFORMATION FOR SEQ ID NO: 6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 999 base                                                           (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "cDNA domains with syntheticc                                                              linker(s)"                                                      -    (vii) IMMEDIATE SOURCE:                                                   #insert of Fv.4715-mycindIII-EcoRI                                             -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:40..468                                                           (D) OTHER INFORMATION:/pro - #duct= "pelB-VH4715"                    -     (ix) FEATURE:                                                                      (A) NAME/KEY: sig.sub.-- - #peptide                                            (B) LOCATION:40..105                                                           (D) OTHER INFORMATION:/pro - #duct= "pectate lyase"                  -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:106..465                                                          (D) OTHER INFORMATION:/pro - #duct= "VH4715"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:520..963                                                          (D) OTHER INFORMATION:/pro - #duct= "pelB-VL4715-myc"                -     (ix) FEATURE:                                                                      (A) NAME/KEY: sig.sub.-- - #peptide                                            (B) LOCATION:520..585                                                          (D) OTHER INFORMATION:/pro - #duct= "pectate lyase"                  -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:586..927                                                          (D) OTHER INFORMATION:/pro - #duct= "VL4715"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: misc RNA                                                         (B) LOCATION:928..960                                                          (D) OTHER INFORMATION:/pro - #duct= "myc-tag"                        #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC - # CTA TTG             54                                                                           #       Met Lys Tyr Leu Leu                                                    20                                                                             - CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GC - #T GCC CAA CCA GCG ATG           102                                                                           Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Al - #a Ala Gln Pro Ala Met            5                                                                              - GCC CAG GTG CAG CTG CAG GAG TCA GGG GGA GA - #C TTA GTG AAG CCT GGA           150                                                                           Ala Gln Val Gln Leu Gln Glu Ser Gly Gly As - #p Leu Val Lys Pro Gly            #  15                                                                          - GGG TCC CTG ACA CTC TCC TGT GCA ACC TCT GG - #A TTC ACT TTC AGT AGT           198                                                                           Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gl - #y Phe Thr Phe Ser Ser            #                30                                                            - TAT GCC TTT TCT TGG GTC CGC CAG ACC TCA GA - #C AAG AGT CTG GAG TGG           246                                                                           Tyr Ala Phe Ser Trp Val Arg Gln Thr Ser As - #p Lys Ser Leu Glu Trp            #            45                                                                - GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT AC - #C TAT TAT TCA GAC AAT           294                                                                           Val Ala Thr Ile Ser Ser Thr Asp Thr Tyr Th - #r Tyr Tyr Ser Asp Asn            #        60                                                                    - GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AA - #T GGC AAG AAC ACC CTG           342                                                                           Val Lys Gly Arg Phe Thr Ile Ser Arg Asp As - #n Gly Lys Asn Thr Leu            #    75                                                                        - TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GA - #C ACA GCC GTG TAT TAC           390                                                                           Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu As - #p Thr Ala Val Tyr Tyr            #95                                                                            - TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TA - #T TTT GAC TAC TGG GGC           438                                                                           Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Ty - #r Phe Asp Tyr Trp Gly            #               110                                                            - CAA GGG ACC ACG GTC ACC GTC TCC TCA TAA TA - #AGAGCTAT GGGAGCTTGC             488                                                                           Gln Gly Thr Thr Val Thr Val Ser Ser                                            #           120                                                                #TTG CCT ACG       540G AGACAGTCAT A ATG AAA TAC CTA                           #                 Met - # Lys Tyr Leu Leu Pro Thr                              20 - #                                                                         - GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CA - #A CCA GCG ATG GCC GAC           588                                                                           Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gl - #n Pro Ala Met Ala Asp            #1#5                                                                           - ATC GAG CTC ACT CAG TCT CCA TTC TCC CTG AC - #T GTG ACA GCA GGA GAG           636                                                                           Ile Glu Leu Thr Gln Ser Pro Phe Ser Leu Th - #r Val Thr Ala Gly Glu            #             15                                                               - AAG GTC ACT ATG AAT TGC AAG TCC GGT CAG AG - #T CTG TTA AAC AGT GTA           684                                                                           Lys Val Thr Met Asn Cys Lys Ser Gly Gln Se - #r Leu Leu Asn Ser Val            #        30                                                                    - AAT CAG AGG AAC TAC TTG ACC TGG TAC CAG CA - #G AAG CCA GGG CAG CCT           732                                                                           Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gl - #n Lys Pro Gly Gln Pro            #    45                                                                        - CCT AAA CTG TTG ATC TAC TGG GCA TCC ACT AG - #G GAA TCT GGA GTC CCT           780                                                                           Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Ar - #g Glu Ser Gly Val Pro            #65                                                                            - GAT CGC TTC ACA GCC AGT GGA TCT GGA ACA GA - #T TTC ACT CTC ACC ATC           828                                                                           Asp Arg Phe Thr Ala Ser Gly Ser Gly Thr As - #p Phe Thr Leu Thr Ile            #                80                                                            - AGC AGT GTG CAG GCT GAA GAC CTG GCA GTT TA - #T TAC TGT CAG AAT GAT           876                                                                           Ser Ser Val Gln Ala Glu Asp Leu Ala Val Ty - #r Tyr Cys Gln Asn Asp            #            95                                                                - TAT ACT TAT CCG TTC ACG TTC GGA GGG GGG AC - #C AAG CTC GAG ATC AAA           924                                                                           Tyr Thr Tyr Pro Phe Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys            #       110                                                                    - CGG GAA CAA AAA CTC ATC TCA GAA GAG GAT CT - #G AAT TAA TAAGATCAAA            973                                                                           Arg Glu Gln Lys Leu Ile Ser Glu Glu Asp Le - #u Asn                            #   125                                                                        #             999  GCTC GAATTC                                                 - (2) INFORMATION FOR SEQ ID NO: 7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 142 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl - #y Leu Leu Leu Leu Ala          10                                                                             - Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gl - #n Glu Ser Gly Gly Asp          #  10                                                                          - Leu Val Lys Pro Gly Gly Ser Leu Thr Leu Se - #r Cys Ala Thr Ser Gly          #                25                                                            - Phe Thr Phe Ser Ser Tyr Ala Phe Ser Trp Va - #l Arg Gln Thr Ser Asp          #            40                                                                - Lys Ser Leu Glu Trp Val Ala Thr Ile Ser Se - #r Thr Asp Thr Tyr Thr          #        55                                                                    - Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Th - #r Ile Ser Arg Asp Asn          #    70                                                                        - Gly Lys Asn Thr Leu Tyr Leu Gln Met Ser Se - #r Leu Lys Ser Glu Asp          #90                                                                            - Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Ty - #r Tyr Gly Lys Gly Tyr          #                105                                                           - Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser                  #           120                                                                - (2) INFORMATION FOR SEQ ID NO: 8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 147 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl - #y Leu Leu Leu Leu Ala          10                                                                             - Ala Gln Pro Ala Met Ala Asp Ile Glu Leu Th - #r Gln Ser Pro Phe Ser          #  10                                                                          - Leu Thr Val Thr Ala Gly Glu Lys Val Thr Me - #t Asn Cys Lys Ser Gly          #                25                                                            - Gln Ser Leu Leu Asn Ser Val Asn Gln Arg As - #n Tyr Leu Thr Trp Tyr          #            40                                                                - Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Le - #u Ile Tyr Trp Ala Ser          #        55                                                                    - Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Th - #r Ala Ser Gly Ser Gly          #    70                                                                        - Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gl - #n Ala Glu Asp Leu Ala          #90                                                                            - Val Tyr Tyr Cys Gln Asn Asp Tyr Thr Tyr Pr - #o Phe Thr Phe Gly Gly          #                105                                                           - Gly Thr Lys Leu Glu Ile Lys Arg Glu Gln Ly - #s Leu Ile Ser Glu Glu          #           120                                                                - Asp Leu Asn                                                                          125                                                                    - (2) INFORMATION FOR SEQ ID NO: 9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 924 base                                                           (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "cDNA domains with syntheticc                                                              linker(s)"                                                      -    (vii) IMMEDIATE SOURCE:                                                   #insert of scFv.4715-mycdIII-EcoRI                                             -     (ix) FEATURE:                                                                      (A) NAME/KEY: sig.sub.-- - #peptide                                            (B) LOCATION:40..105                                                           (D) OTHER INFORMATION:/pro - #duct= "pectate lyase"                  -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:106..465                                                          (D) OTHER INFORMATION:/pro - #duct= "VH4715"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: misc.sub.-- - #RNA                                               (B) LOCATION:466..510                                                          (D) OTHER INFORMATION:/pro - #duct= "(Gly4Ser)3-linker"              -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:511..852                                                          (D) OTHER INFORMATION:/pro - #duct= "VL4715"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: misc.sub.-- - #RNA                                               (B) LOCATION:853..885                                                          (D) OTHER INFORMATION:/pro - #duct= "myc-tag"                        -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:40..888                                                           (D) OTHER INFORMATION:/pro - #duct= "pelB-VH4715-(Gly4Ser)3-                        VL4715-myc"                                                     #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC - # CTA TTG             54                                                                           #       Met Lys Tyr Leu Leu                                                    20                                                                             - CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GC - #T GCC CAA CCA GCG ATG           102                                                                           Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Al - #a Ala Gln Pro Ala Met            5                                                                              - GCC CAG GTG CAG CTG CAG GAG TCA GGG GGA GA - #C TTA GTG AAG CCT GGA           150                                                                           Ala Gln Val Gln Leu Gln Glu Ser Gly Gly As - #p Leu Val Lys Pro Gly            #  15                                                                          - GGG TCC CTG ACA CTC TCC TGT GCA ACC TCT GG - #A TTC ACT TTC AGT AGT           198                                                                           Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gl - #y Phe Thr Phe Ser Ser            #                30                                                            - TAT GCC TTT TCT TGG GTC CGC CAG ACC TCA GA - #C AAG AGT CTG GAG TGG           246                                                                           Tyr Ala Phe Ser Trp Val Arg Gln Thr Ser As - #p Lys Ser Leu Glu Trp            #            45                                                                - GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT AC - #C TAT TAT TCA GAC AAT           294                                                                           Val Ala Thr Ile Ser Ser Thr Asp Thr Tyr Th - #r Tyr Tyr Ser Asp Asn            #        60                                                                    - GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AA - #T GGC AAG AAC ACC CTG           342                                                                           Val Lys Gly Arg Phe Thr Ile Ser Arg Asp As - #n Gly Lys Asn Thr Leu            #    75                                                                        - TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GA - #C ACA GCC GTG TAT TAC           390                                                                           Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu As - #p Thr Ala Val Tyr Tyr            #95                                                                            - TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TA - #T TTT GAC TAC TGG GGC           438                                                                           Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Ty - #r Phe Asp Tyr Trp Gly            #               110                                                            - CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GG - #A GGC GGT TCA GGC GGA           486                                                                           Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gl - #y Gly Gly Ser Gly Gly            #           125                                                                - GGT GGC TCT GGC GGT GGC GGA TCG GAC ATC GA - #G CTC ACT CAG TCT CCA           534                                                                           Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gl - #u Leu Thr Gln Ser Pro            #       140                                                                    - TTC TCC CTG ACT GTG ACA GCA GGA GAG AAG GT - #C ACT ATG AAT TGC AAG           582                                                                           Phe Ser Leu Thr Val Thr Ala Gly Glu Lys Va - #l Thr Met Asn Cys Lys            #   155                                                                        - TCC GGT CAG AGT CTG TTA AAC AGT GTA AAT CA - #G AGG AAC TAC TTG ACC           630                                                                           Ser Gly Gln Ser Leu Leu Asn Ser Val Asn Gl - #n Arg Asn Tyr Leu Thr            160                 1 - #65                 1 - #70                 1 -        #75                                                                            - TGG TAC CAG CAG AAG CCA GGG CAG CCT CCT AA - #A CTG TTG ATC TAC TGG           678                                                                           Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Ly - #s Leu Leu Ile Tyr Trp            #               190                                                            - GCA TCC ACT AGG GAA TCT GGA GTC CCT GAT CG - #C TTC ACA GCC AGT GGA           726                                                                           Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Ar - #g Phe Thr Ala Ser Gly            #           205                                                                - TCT GGA ACA GAT TTC ACT CTC ACC ATC AGC AG - #T GTG CAG GCT GAA GAC           774                                                                           Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Se - #r Val Gln Ala Glu Asp            #       220                                                                    - CTG GCA GTT TAT TAC TGT CAG AAT GAT TAT AC - #T TAT CCG TTC ACG TTC           822                                                                           Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Th - #r Tyr Pro Phe Thr Phe            #   235                                                                        - GGA GGG GGG ACC AAG CTC GAG ATC AAA CGG GA - #A CAA AAA CTC ATC TCA           870                                                                           Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gl - #u Gln Lys Leu Ile Ser            240                 2 - #45                 2 - #50                 2 -        #55                                                                            - GAA GAG GAT CTG AAT TAA TAAGATCAAA CGGTAATAAG GA - #TCCAGCTC GAATTC           924                                                                           Glu Glu Asp Leu Asn                                                                            260                                                            - (2) INFORMATION FOR SEQ ID NO: 10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 282 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl - #y Leu Leu Leu Leu Ala          10                                                                             - Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gl - #n Glu Ser Gly Gly Asp          #  10                                                                          - Leu Val Lys Pro Gly Gly Ser Leu Thr Leu Se - #r Cys Ala Thr Ser Gly          #                25                                                            - Phe Thr Phe Ser Ser Tyr Ala Phe Ser Trp Va - #l Arg Gln Thr Ser Asp          #            40                                                                - Lys Ser Leu Glu Trp Val Ala Thr Ile Ser Se - #r Thr Asp Thr Tyr Thr          #        55                                                                    - Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Th - #r Ile Ser Arg Asp Asn          #    70                                                                        - Gly Lys Asn Thr Leu Tyr Leu Gln Met Ser Se - #r Leu Lys Ser Glu Asp          #90                                                                            - Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Ty - #r Tyr Gly Lys Gly Tyr          #                105                                                           - Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Gly Gly          #           120                                                                - Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gl - #y Gly Ser Asp Ile Glu          #       135                                                                    - Leu Thr Gln Ser Pro Phe Ser Leu Thr Val Th - #r Ala Gly Glu Lys Val          #   150                                                                        - Thr Met Asn Cys Lys Ser Gly Gln Ser Leu Le - #u Asn Ser Val Asn Gln          155                 1 - #60                 1 - #65                 1 -        #70                                                                            - Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pr - #o Gly Gln Pro Pro Lys          #               185                                                            - Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Se - #r Gly Val Pro Asp Arg          #           200                                                                - Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Th - #r Leu Thr Ile Ser Ser          #       215                                                                    - Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cy - #s Gln Asn Asp Tyr Thr          #   230                                                                        - Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Le - #u Glu Ile Lys Arg Glu          235                 2 - #40                 2 - #45                 2 -        #50                                                                            - Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn                                      #               260                                                            - (2) INFORMATION FOR SEQ ID NO: 11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 1706 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "cDNA domains with syntheticc                                                              linker(s)"                                                      -    (vii) IMMEDIATE SOURCE:                                                   #insert of pGOSA.EE: HindIII-EcoRI                                             -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:40..864                                                           (D) OTHER INFORMATION:/pro - #duct= "pelB-VH4715-LiA-VH3418"         -     (ix) FEATURE:                                                                      (A) NAME/KEY: sig.sub.-- - #peptide                                            (B) LOCATION:40..105                                                           (D) OTHER INFORMATION:/pro - #duct= "pectate lyase"                  -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:106..465                                                          (D) OTHER INFORMATION:/pro - #duct= "VH4715"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: misc.sub.-- - #RNA                                               (B) LOCATION:466..522                                                          (D) OTHER INFORMATION:/pro - #duct= "linkerA (Gly4Ser)                              3AlaGlySerAl - #a"                                              -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:523..861                                                          (D) OTHER INFORMATION:/pro - #duct= "VH3418"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:913..1689                                                         (D) OTHER INFORMATION:/pro - #duct= "pelB-VL3418-LiV-VL4715"         -     (ix) FEATURE:                                                                      (A) NAME/KEY: sig.sub.-- - #peptide                                            (B) LOCATION:913..978                                                          (D) OTHER INFORMATION:/pro - #duct= "pectate lyase"                  -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:979..1299                                                         (D) OTHER INFORMATION:/pro - #duct= "VL3418"                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: misc.sub.-- - #RNA                                               (B) LOCATION:1300..1344                                                        (D) OTHER INFORMATION:/pro - #duct= "linker V (Gly4Ser)2Gly4Val"     -     (ix) FEATURE:                                                                      (A) NAME/KEY: mat.sub.-- - #peptide                                            (B) LOCATION:1345..1686                                                        (D) OTHER INFORMATION:/pro - #duct= "VL4715"                         #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - AAGCTTGCAT GGAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC - # CTA TTG             54                                                                           #       Met Lys Tyr Leu Leu                                                    20                                                                             - CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GC - #T GCC CAA CCA GCG ATG           102                                                                           Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Al - #a Ala Gln Pro Ala Met            5                                                                              - GCC CAG GTG CAG CTG CAG GAG TCA GGG GGA GA - #C TTA GTG AAG CCT GGA           150                                                                           Ala Gln Val Gln Leu Gln Glu Ser Gly Gly As - #p Leu Val Lys Pro Gly            #  15                                                                          - GGG TCC CTG ACA CTC TCC TGT GCA ACC TCT GG - #A TTC ACT TTC AGT AGT           198                                                                           Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gl - #y Phe Thr Phe Ser Ser            #                30                                                            - TAT GCC TTT TCT TGG GTC CGC CAG ACC TCA GA - #C AAG AGT CTG GAG TGG           246                                                                           Tyr Ala Phe Ser Trp Val Arg Gln Thr Ser As - #p Lys Ser Leu Glu Trp            #            45                                                                - GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT AC - #C TAT TAT TCA GAC AAT           294                                                                           Val Ala Thr Ile Ser Ser Thr Asp Thr Tyr Th - #r Tyr Tyr Ser Asp Asn            #        60                                                                    - GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AA - #T GGC AAG AAC ACC CTG           342                                                                           Val Lys Gly Arg Phe Thr Ile Ser Arg Asp As - #n Gly Lys Asn Thr Leu            #    75                                                                        - TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GA - #C ACA GCC GTG TAT TAC           390                                                                           Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu As - #p Thr Ala Val Tyr Tyr            #95                                                                            - TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TA - #T TTT GAC TAC TGG GGC           438                                                                           Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Ty - #r Phe Asp Tyr Trp Gly            #               110                                                            - CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GG - #A GGC GGT TCA GGC GGA           486                                                                           Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gl - #y Gly Gly Ser Gly Gly            #           125                                                                - GGT GGC TCT GGC GGT GGC GGA TCG GCC GGT TC - #G GCC CAG GTC CAG CTG           534                                                                           Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Se - #r Ala Gln Val Gln Leu            #       140                                                                    - CAA CAG TCA GGA CCT GAG CTG GTA AAG CCT GG - #G GCT TCA GTG AAG ATG           582                                                                           Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Met            #   155                                                                        - TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT AG - #C TAT GTT ATG CAC TGG           630                                                                           Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Se - #r Tyr Val Met His Trp            160                 1 - #65                 1 - #70                 1 -        #75                                                                            - GTG AAA CAG AAG CCT GGG CAG GGC CTT GAG TG - #G ATT GGA TAT ATT TAT           678                                                                           Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Tr - #p Ile Gly Tyr Ile Tyr            #               190                                                            - CCT TAC AAT GAT GGT ACT AAG TAC AAT GAG AA - #G TTC AAA GGC AAG GCC           726                                                                           Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Ly - #s Phe Lys Gly Lys Ala            #           205                                                                - ACA CTG ACT TCA GAC AAA TCC TCC AGC ACA GC - #C TAC ATG GAG CTC AGC           774                                                                           Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Al - #a Tyr Met Glu Leu Ser            #       220                                                                    - AGC CTG ACC TCT GAG GAC TCT GCG GTC TAT TA - #C TGT TCA AGA CGC TTT           822                                                                           Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ty - #r Cys Ser Arg Arg Phe            #   235                                                                        - GAC TAC TGG GGC CAA GGG ACC ACC GTC ACC GT - #C TCC TCA TAA                  # 864                                                                          Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Va - #l Ser Ser                        240                 2 - #45                 2 - #50                            #AAA TAC     921CTGCATG CAAATTCTAT TTCAAGGAGA CAGTCATA ATG                     #                 Met - # Lys Tyr                                              20 - #                                                                         - CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TT - #A CTC GCT GCC CAA CCA           969                                                                           Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Le - #u Leu Ala Ala Gln Pro            5                                                                              - GCG ATG GCC GAC ATC GAG CTC ACC CAG TCT CC - #A TCT TCC ATG TAT GCA          1017                                                                           Ala Met Ala Asp Ile Glu Leu Thr Gln Ser Pr - #o Ser Ser Met Tyr Ala            #          10                                                                  - TCT CTA GGA GAG AGA ATC ACT ATC ACT TGC AA - #G GCG AGT CAG GAC ATT          1065                                                                           Ser Leu Gly Glu Arg Ile Thr Ile Thr Cys Ly - #s Ala Ser Gln Asp Ile            #    25                                                                        - AAT ACC TAT TTA ACC TGG TTC CAG CAG AAA CC - #A GGG AAA TCT CCC AAG          1113                                                                           Asn Thr Tyr Leu Thr Trp Phe Gln Gln Lys Pr - #o Gly Lys Ser Pro Lys            #45                                                                            - ACC CTG ATC TAT CGT GCA AAC AGA TTG CTA GA - #T GGG GTC CCA TCA AGG          1161                                                                           Thr Leu Ile Tyr Arg Ala Asn Arg Leu Leu As - #p Gly Val Pro Ser Arg            #                60                                                            - TTC AGT GGC AGT GGA TCT GGG CAA GAT TAT TC - #T CTC ACC ATC AGC AGC          1209                                                                           Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Se - #r Leu Thr Ile Ser Ser            #            75                                                                - CTG GAC TAT GAA GAT ATG GGA ATT TAT TAT TG - #T CTA CAA TAT GAT GAG          1257                                                                           Leu Asp Tyr Glu Asp Met Gly Ile Tyr Tyr Cy - #s Leu Gln Tyr Asp Glu            #        90                                                                    - TTG TAC ACG TTC GGA GGG GGG ACC AAG CTC GA - #G ATC AAA CGG GGT GGA          1305                                                                           Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Gl - #u Ile Lys Arg Gly Gly            #    105                                                                       - GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GG - #C GGA GTC GAC ATC GAA          1353                                                                           Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gl - #y Gly Val Asp Ile Glu            110                 1 - #15                 1 - #20                 1 -        #25                                                                            - CTC ACT CAG TCT CCA TTC TCC CTG ACT GTG AC - #A GCA GGA GAG AAG GTC          1401                                                                           Leu Thr Gln Ser Pro Phe Ser Leu Thr Val Th - #r Ala Gly Glu Lys Val            #               140                                                            - ACT ATG AAT TGC AAG TCC GGT CAG AGT CTG TT - #A AAC AGT GTA AAT CAG          1449                                                                           Thr Met Asn Cys Lys Ser Gly Gln Ser Leu Le - #u Asn Ser Val Asn Gln            #           155                                                                - AGG AAC TAC TTG ACC TGG TAC CAG CAG AAG CC - #A GGG CAG CCT CCT AAA          1497                                                                           Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pr - #o Gly Gln Pro Pro Lys            #       170                                                                    - CTG TTG ATC TAC TGG GCA TCC ACT AGG GAA TC - #T GGA GTC CCT GAT CGC          1545                                                                           Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Se - #r Gly Val Pro Asp Arg            #   185                                                                        - TTC ACA GCC AGT GGA TCT GGA ACA GAT TTC AC - #T CTC ACC ATC AGC AGT          1593                                                                           Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Th - #r Leu Thr Ile Ser Ser            190                 1 - #95                 2 - #00                 2 -        #05                                                                            - GTG CAG GCT GAA GAC CTG GCA GTT TAT TAC TG - #T CAG AAT GAT TAT ACT          1641                                                                           Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cy - #s Gln Asn Asp Tyr Thr            #               220                                                            - TAT CCG TTC ACG TTC GGA GGG GGG ACC AAG CT - #C GAA ATC AAA CGG TAA          1689                                                                           Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Le - #u Glu Ile Lys Arg                #           235                                                                # 1706             C                                                           - (2) INFORMATION FOR SEQ ID NO: 12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 274 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl - #y Leu Leu Leu Leu Ala          10                                                                             - Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gl - #n Glu Ser Gly Gly Asp          #  10                                                                          - Leu Val Lys Pro Gly Gly Ser Leu Thr Leu Se - #r Cys Ala Thr Ser Gly          #                25                                                            - Phe Thr Phe Ser Ser Tyr Ala Phe Ser Trp Va - #l Arg Gln Thr Ser Asp          #            40                                                                - Lys Ser Leu Glu Trp Val Ala Thr Ile Ser Se - #r Thr Asp Thr Tyr Thr          #        55                                                                    - Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Th - #r Ile Ser Arg Asp Asn          #    70                                                                        - Gly Lys Asn Thr Leu Tyr Leu Gln Met Ser Se - #r Leu Lys Ser Glu Asp          #90                                                                            - Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Ty - #r Tyr Gly Lys Gly Tyr          #                105                                                           - Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Th - #r Val Ser Ser Gly Gly          #           120                                                                - Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gl - #y Gly Ser Ala Gly Ser          #       135                                                                    - Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gl - #u Leu Val Lys Pro Gly          #   150                                                                        - Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gl - #y Tyr Thr Phe Thr Ser          155                 1 - #60                 1 - #65                 1 -        #70                                                                            - Tyr Val Met His Trp Val Lys Gln Lys Pro Gl - #y Gln Gly Leu Glu Trp          #               185                                                            - Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Th - #r Lys Tyr Asn Glu Lys          #           200                                                                - Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Ly - #s Ser Ser Ser Thr Ala          #       215                                                                    - Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu As - #p Ser Ala Val Tyr Tyr          #   230                                                                        - Cys Ser Arg Arg Phe Asp Tyr Trp Gly Gln Gl - #y Thr Thr Val Thr Val          235                 2 - #40                 2 - #45                 2 -        #50                                                                            - Ser Ser                                                                      - (2) INFORMATION FOR SEQ ID NO: 13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 258 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl - #y Leu Leu Leu Leu Ala          10                                                                             - Ala Gln Pro Ala Met Ala Asp Ile Glu Leu Th - #r Gln Ser Pro Ser Ser          #  10                                                                          - Met Tyr Ala Ser Leu Gly Glu Arg Ile Thr Il - #e Thr Cys Lys Ala Ser          #                25                                                            - Gln Asp Ile Asn Thr Tyr Leu Thr Trp Phe Gl - #n Gln Lys Pro Gly Lys          #            40                                                                - Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Ar - #g Leu Leu Asp Gly Val          #        55                                                                    - Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gl - #n Asp Tyr Ser Leu Thr          #    70                                                                        - Ile Ser Ser Leu Asp Tyr Glu Asp Met Gly Il - #e Tyr Tyr Cys Leu Gln          #90                                                                            - Tyr Asp Glu Leu Tyr Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys          #                105                                                           - Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Se - #r Gly Gly Gly Gly Val          #           120                                                                - Asp Ile Glu Leu Thr Gln Ser Pro Phe Ser Le - #u Thr Val Thr Ala Gly          #       135                                                                    - Glu Lys Val Thr Met Asn Cys Lys Ser Gly Gl - #n Ser Leu Leu Asn Ser          #   150                                                                        - Val Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gl - #n Gln Lys Pro Gly Gln          155                 1 - #60                 1 - #65                 1 -        #70                                                                            - Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Th - #r Arg Glu Ser Gly Val          #               185                                                            - Pro Asp Arg Phe Thr Ala Ser Gly Ser Gly Th - #r Asp Phe Thr Leu Thr          #           200                                                                - Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Va - #l Tyr Tyr Cys Gln Asn          #       215                                                                    - Asp Tyr Thr Tyr Pro Phe Thr Phe Gly Gly Gl - #y Thr Lys Leu Glu Ile          #   230                                                                        - Lys Arg                                                                      235                                                                            - (2) INFORMATION FOR SEQ ID NO: 14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 25 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.1                                              #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #               25 AATG GCAAG                                                  - (2) INFORMATION FOR SEQ ID NO: 15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 45 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.2                                              #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #45                GAAC CGGCCGATCC GCCACCGCCA GAGCC                            - (2) INFORMATION FOR SEQ ID NO: 16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 45 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.3                                              #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #45                CGGC CCAGGTCCAG CTGCAACAGT CAGGA                            - (2) INFORMATION FOR SEQ ID NO: 17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 53 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.4                                              #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - CTACATGAAT TCGCTAGCTT ATTATGAGGA GACGGTGACG GTGGTCCCTT GG - #C                 53                                                                           - (2) INFORMATION FOR SEQ ID NO: 18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 36 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.5                                              #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #       36         CTGC ATGCAAATTC TATTTC                                      - (2) INFORMATION FOR SEQ ID NO: 19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 23 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.6                                              #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #                23AACG GGG                                                    - (2) INFORMATION FOR SEQ ID NO: 20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 36 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.7                                              #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #       36         ACTC CGCCACCGCC AGAGCC                                      - (2) INFORMATION FOR SEQ ID NO: 21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 39 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= synthetic DNA"CRIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.8                                              #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #    39            AACT CACTCAGTCT CCATTCTCC                                   - (2) INFORMATION FOR SEQ ID NO: 22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 50 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.9                                              #22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #              50GGCCGC TTATTACCGT TTGATTTCGA GCTTGGTCCC                       - (2) INFORMATION FOR SEQ ID NO: 23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 41 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.1 - #0                                         #23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #   41             CTCC TCACAGGTCC AGTTGCAACA G                                - (2) INFORMATION FOR SEQ ID NO: 24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 44 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.1 - #1                                         #24:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    # 44               AACG GGACATCGAA CTCACTCAGT CTCC                             - (2) INFORMATION FOR SEQ ID NO: 25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 41 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer DBL.1 - #2                                         #25:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #   41             CTCC TCACAGGTGC AGTTGCAGGA G                                - (2) INFORMATION FOR SEQ ID NO: 26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 22 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer PCR.5 - #1                                         #26:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #                 22TCW GG                                                     - (2) INFORMATION FOR SEQ ID NO: 27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 32 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer PCR.8 - #9                                         #27:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #          32      GTGG TCCCTTGGCC CC                                          - (2) INFORMATION FOR SEQ ID NO: 28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 24 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer PCR.9 - #0                                         #28:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #                24AGTC TCCA                                                   - (2) INFORMATION FOR SEQ ID NO: 29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 22 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: other nucleic acid                                   #= "synthetic DNA"RIPTION: /desc                                               -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: primer PCR.1 - #16                                        #29:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #                 22GTC CC                                                     - (2) INFORMATION FOR SEQ ID NO: 30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 13 amino                                                           (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #30:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Thr Thr Val Thr Val Ser Ser Gln Val Gln Le - #u Gln Gln                      #                10                                                            - (2) INFORMATION FOR SEQ ID NO: 31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 12 amino                                                           (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              #31:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Lys Leu Glu Ile Lys Arg Asp Ile Glu Leu Th - #r Gln                          #                10                                                            __________________________________________________________________________ 

We claim:
 1. A bispecific or bivalent double head antibody fragment, which comprises a binding complex containing two polypeptide chains, whereby one polypeptide chain comprises two times a variable domain of a heavy chain (V_(H)) in series and the other polypeptide chain comprises two times a variable domain of a light chain (V_(L)) in series, and the binding complex contains two pairs of variable domains (V_(H) -A/V_(L) -A and V_(H) -B/V_(L) -B), wherein said double head antibody fragments have bifunctional antigen binding activity and said complex compirses one polypeptide chain wherein the variable regions are connected by a peptide linker or neither polypeptide chain is connected by a peptide linker.
 2. An antibody fragment according to claim 1, in which one polypeptide chain comprises a first V_(H) linked to a second V_(H) and the other polypeptide chain comprises a first V_(L) linked to a second V_(L).
 3. An antibody fragment according to claim 2, in which one polypeptide chain comprises a first V_(H) (V_(H) -A) followed by a second V_(H) (V_(H) -B) with or without a connecting peptide linker (Li_(H)), thus, and the other polypeptide chain comprises a first V_(L) (V_(L) -A) preceded by a second V_(L) (V_(L) -B) with or without a connecting peptide linker (Li_(L)), thus provided that the antibody fragment in total comprises only one peptide linker or no peptide linker.
 4. An antibody fragment analogue according to claim 1, in which the two variable domains are from different Fvs resulting in a bispecific antibody fragment.
 5. An antibody fragment according to claim 1, in which the specificities A and B are the same resulting in a bivalent antibody fragment.
 6. A process for producing an antibody fragment according to any one of claims 1, 2 and 3-5, which comprises(1) transforming a Gram negative bacterial host by incorporating into that host a DNA encoding the two V_(H) 's in series with or without a connecting peptide linker and a DNA encoding the two V_(L) 's in series with or without a connecting peptide linker, (2) culturing such transformed host under conditions whereby the linked V_(H) 's and the linked V_(L) 's are formed, and (3) allowing the two linked V_(H) 's and the two connected V_(L) 's to combine to each other under formation of a double head antibody fragment whereby either the V_(H) 's are connected by a peptide linker or the V_(L) 's are connected by a peptide linker or neither V_(H) 's nor V_(L) 's are connected by a peptide linker, and (4) optionally collecting the double head antibody fragment.
 7. A process according to claim 6, in which the host comprises E. coli, and higher eukaryotic organisms or cell cultures thereof.
 8. An immunoassay comprising an antibody fragment as a binding means, the improvement wherein said antibody fragment comprises a double head antibody fragment according to claim
 1. 9. An antibody fragment according to claim 1, comprising a peptide linker, said peptide linker comprising sufficient residues to span 30 to 35 Å. 